Characterization of the protein kinases ERK3 and ERK4 and their interaction with MK5

A thesis submitted for the degree of Doctor of Philosophy (PhD) in the subject of Molecular and cellular Biology by Shashi Kant, M.Sc. Biotechnology November 2006

Hannover Medical School International MD/PhD program “Molecular Medicine” Institutes of Biochemistry

Acknowledged by the MD/PhD committee and head of Hannover Medical School

President:

Prof. Dr. med. D. Bitter-Suermann

Supervisor:

Prof. Dr. Matthias Gaestel

Co-supervisors:

Prof. Dr. Dietmar Manstein Prof. Dr. Thomas F Schulz Dr. Ole-Morten Seternes,

External expert:

(University of Tromsoe)

Internal expert:

Dr. Andreas Kispert

Day of final exam/public defense:

10th November 2006

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Table of Contents I INTRODUCTION...................................................................................... 4 I-1. MAPK signaling pathways....................................................................... 5 I-1.1. Mitogen activated ERK1/2 kinase cascades.......................................... 7 I-1.2. The Stress-activated JNK- and p38 MAP signaling pathways.............. 8 I-1.2.1. JNK-MAP kinase cascade....................................................................8 I-1.2.2. p38 MAP kinase cascade..................................................................... 9 I-1.2.3. Physiological roles of JNK- and p38 MAPK cascades...................... 10 I-1.3. ERK5 kinase cascade……….................................................................11 I-1.4. ERK7/8 signaling pathways……….......................................................12 I-1.5. ERK3/4 signaling pathways………...................................................... 12 I-1.5.1. ERK3/4 identification and structure................................................... 12 I-1.5.2 ERK3 kinase cascade........................................................................... 13 I-1.5.3 ERK4 kinase cascade …………………………….......................……15 I-1.6 MAPKAP kinase cascades……………………...................……………15 I-1.7 MAP Kinase phosphatase………………………................................…17 II RESULTS................................................................................................... 18 II-1. Scaffolding by ERK3 regulates MK5 in development.............................19 II-2. Characterization of the atypical MAP kinase ERK4 and its activation of the MAPK-activated protein kinase MK5....................................................33 III SUMMARY AND DISCUSSION........................................................... 46 IV REFERENCES......................................................................................... 52 ABBREVIATION.......................................................................................... 63 ACKNOWLEDGMENTS..............................................................................66 CURRICULUM VITAE.................................................................................68 DECLARATION………………………………………………………….....72

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I. INTRODUCTION

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INTRODUCTION I-1. MAPK signaling pathways In nature, there is always a beautiful balance between the autonomy and dependence on surrounding and that’s even present at cellular level. Cell responds to any changes in their surrounding environment by switching on or off its intracellular programs. Such a response needs to be quick, specific, spatially and temporally co-ordinated with the signals received from other cells or tissues, in order to induce an appropriate response to all these challenges. There are several pathways in cells that can be activated by different stimuli from the environment such as mitogen or growth factors, cellular stresses like UV, osmotic stress and oxidative stress (for review see (Platanias et al. 2003)). Mitogen-activated protein kinases (MAPKs) are also known as extra-cellular regulated kinases (ERKs) and their well characterized signaling pathways are among the most widespread mechanisms of eukaryotic cell regulations. MAPKs represent a family of evolutionary highly conserved enzymes. All eukaryotic cells possess multiple MAPK cascades, each of which is preferentially recruited by distinct sets of stimuli, thereby allowing cells to respond coordinately to multiple divergent inputs (Rubinfeld et al. 2005). Development is a complex process, implying many signaling cascades that act in an orchestrated manner and the challenge is to understand the molecular mechanisms, which govern these processes. MAPKs are involved in developmental processes, and a classical example of MAPK is the sevenless or boss mutant of Drosophila eye development (Tomlinson et al. 2001). Other major pathways involved in development are Wnt, Hedgehog and Notch and are considered as backbone of signaling in development (for review see (Pires-daSilva et al. 2003)). In brief, Wnt pathway is activated by binding of extra-cellular Wnt proteins to Frizzled receptors, what is important for activation of cytosolic disheveled protein. Active disheveled inactivates GSK-3β by phosphorylation and this leads to dissociation of GSK-3β from complex that contains Axin, APC and β-catenin. Thus GSK-3β-mediated phosphorylation and consequent degradation of β-catenin is prevented and β-catenin is accumulated in the nucleus leading to activation of TCF/LEF transcription factors. The Wnt signaling pathway controls cell proliferation and body patterning throughout development processes from flies to mammals. Wnt signaling pathway regulates axis specification as well as stem cell proliferation and is also linked to different human cancers (for review see (Willert et al. 2006)). 5

Hedgehog protein binds to Patched receptor that acts as negative regulator for Smoothened receptor. The binding of Hedgehog facilitates the Smoothened receptor to transmit a signal to transcription factors called Gli (Ci in Drosophila). When inactive, Gli or Ci forms a complex with a kinase Fused and a microtubule binding protein Coastal. In absence of signals complex binds to tubulin and Ci is cleaved to generate a transcriptional repressor (Ci75). Activation of this pathway leads the dissociation of complex from microtubules and translocation of full length Ci to nucleus where it functions as a transcription factor (for review see (Wilson et al. 2006)). Notch-Delta pathway is critical for many important processes during embryogenesis. Delta trans-membrane ligand located on the surface of neighboring cell, binds to Notch receptor on target cell that leads to proteolyic cleavage of Notch and the intra-cellular domain of Notch translocates to the nucleus activating transcription factor Su (H)/CSL. The classical role of Notch signaling, for instance in the movement of cells involved in somite segmentation or angiogenesis has been defined (for review see (Katsube et al. 2005)). Involvement of MAPKs into both, prenatal and postnatal regulation of growth and proliferation poses many questions about interplay between MAPKs pathway and other signal transduction cascades that define fate of the cell. MAPKs connect cell surface receptors to critical regulatory targets within cell. A wide variety of extra-cellular stimuli serve as activators of MAPKs pathways leading to appropriate responses of cells, such as proliferation, differentiation, growth and migration (for review see (Kolch 2005; Pearson et al. 2001)) . MAPKs comprise four well defined groups i.e. ERK1/2 (Baulton et al. 1991), JNKs, p38s and ERK5 (BMK) (Zhou et al 1995) as well as some additional members including ERK3 (Baulton et al. 1991), ERK4 (p63mapk, ERK3-related, ERK3β, MAPK4, Prkm4) (Gonzalez et al. 1992), ERK7 (Abe et al. 1999) and its human homolog ERK8 (Abe et al. 2002) have been identifies as a member of this family. MAPK pathways generally have a four tiers kinase module architecture by which the signal is transmitted from an upstream kinase to a downstream kinase by sequential phosphorylation in cells (Figure-1). MAPKs are activated through a specific dual phosphorylation motif located between sub-domain VII and VIII of the catalytic domain. This motif is generally conserved among MAPKs and possesses both threonine and tyrosine residues (TXY) as phosphorylation site (Figure-2). TXY motif confers the specificity to each MAPKs subgroup and allows their independent regulation by MAPK kinases (MKKs). These dual specific protein kinases (MKKs) are relatively specific for each subgroup of MAPKs. As each MKK can be activated by several MAPK kinase kinases (MKKKs), it makes these pathways more 6

complex and diverse (Figure-1). Although MAPKs are the conversion point of there own pathways when followed by several substrate kinases (MAPKAP Kinases or MKs) as the fourth tier of each pathway, the regulation of different activities at cellular level is provided.

I-1.1. Mitogen activated ERK1/2 kinase cascades The ERK1/2 pathway follows the Raf/MEK/ERK module and is strongly activated by mitogenic stimuli such as hormones and growth factors. This pathway transduces signals leading to growth or differentiation of cells. Binding of a growth factor, such as epidermal growth factor (EGF), to the corresponding receptor tyrosine kinase (RTK) leads to its dimerization or oligomerization, and subsequent auto-phosphorylation in trans.

MAPK signaling pathways Growth factors Mitogens

Raf, Mos, Tpl2

MKK1/2

ERK1/2

Elk1, p85, RSK1-4, MNK1/2, MSK1/2

„Stress“ (UV, heat shock, hyperosmolarity, anisomycin, LPS, TNFα, IL1, fMLP...)

MEKKs, MLKs,TAK1, ASK1

Growth factors

MEKK4, MLK3,TAK1, ASK1

MEKK2/3

MKK4/7

MKK3/6

MKK5

JNK1/2/3

p38α/β/γ/δ

ERK5

Shc, p53, NFAT4, c-Jun, ATF2, RNPK

MNK1/2, MEF2, CHOP10, Elk-1, MSK1/2, MAPKAP-K2/3, eEF2K

Mef2A

Figure 1: A MAPK pathways: MAPK pathways generally compose of a four tiers of sequentially activating protein kinase module, in which the signal is transmitted from an upstream kinase to a downstream kinase by sequential phosphorylation in cells leading to different cell responses. Mitogen and cellular stresses lead to activation of the all the four groups of MAPKs. (modified from Raux et al 2004)

Phosphorylated tyrosine residues on the intracellular part of the receptor serve as docking sites for the Src homology (SH2)-domain of the adaptor protein Grb2, which itself binds via 7

its SH3-domain to the proline rich motif of the GDP-GTP exchange factor Sos (son of sevenless). Sos interacts with Ras and activates via exchange of GDP to GTP in Ras. Activated Ras leads to the recruitment of a cytosolic serine/threonine kinase Raf to the cell membrane. Raf is a membrane shuttle kinase and possesses oncogenic properties (Leevers et at. 1994; Avruch et at. 1994; Rapp et al. 1983). The translocation results in the activation of the membrane associated Raf. However, Ras-Raf interaction alone is not sufficient to activate Raf completely (Daum et al. 1994; Dent et al. 1995). Instead, Raf regulation is a complex process, which involves protein-protein interactions, phosphorylation of tyrosine, threonine and serine residues and cellular relocalization (for review see (Kolch 2005)). The first characterized substrate of Raf isoenzymes was the dual specificity kinase MEK or MKK (Macdonald et al. 1993). Once activated, MEK transduces the signals through phosphorylation on TEY motifs in the MAP-kinases ERK1 (p44) and ERK2 (p42) (Boulton et al. 1991; Thomas et al. 1992). The activation of MEK1 and ERK1 through Raf can be enhanced via interaction with the scaffold protein MP1 (MEK partner 1) (Whitmarsh et al. 1998). ERK phosphorylation increases its catalytic activity, mediates oligomerization and alleviates the shuttling of the kinase to the nucleus. ERKs are nuclear shuttle kinases and have several described substrates in contrast to its upstream activators Raf and MEK. Their target proteins are e.g. serine/threonine kinases like ribosomal S6 kinase (RSK1p90) also known as MAPKAPKinase1; RNA-polymerase I, phospholipase A2 and several transcription factors like, Elk-1 and c-Jun (Daum et al. 1994; Blenis et al. 1993; Hill et al. 1995). ERKs are essential elements of mitogenic signaling. Prolonged activation and nuclear retention of ERKs is required for transcription of the cyclin D1 gene (Lavoie et al. 1996), suggesting a mechanism of ERK-mediated enhancement of cell cycle entry.

I-1.2. The Stress-activated JNK- and p38 MAP signaling pathways

I-1.2.1. JNK-MAP kinase cascade There are two different stress induced pathways (i) c-Jun NH2-terminal Kinase (JNK) and (ii) p38 MAP Kinase. JNK family possesses three members they are JNK1, JNK2, and JNK3 (also known as SAPKγ, SAPKα, and SAPKβ, respectively). The first member of the JNK family was originally isolated from rat livers injected with cycloheximide (Kyriakis et al. 1990). JNKs exist in 10 or more different spliced forms and are ubiquitously expressed, although JNK3 is present primarily in the brain. The activation of this pathway is mediated by different stresses and chemical stimuli leads to phosphorylation of numerous protein 8

kinases, which function as MKKKs (MAP Kinase Kinase Kinase). Those MKKKs such as: MEKK1, MEKK2 and MEKK3 (reviewed in (Pearson et al. 2001; Ip et al. 1998)); SPRK/MLK3 (SH3-domain-containing proline-rich kinase/mixed lineage kinase 3) (Rana et al. 1996); or protein kinase Tpl-2 (tumor progression locus 2) and its human homolog Cot (cancer Osaka thyroid) (Hogemann et al. 1999) were implicated in the activation of the JNK pathway. These MKKKs phosphorylate and activate preferentially two MKKs (MAP kinase kinase) MKK4 and MKK7 (MAP kinase kinase), which both reveal homology to MEK (Sanchez et al. 1994). They in turn activate JNK by dual phosphorylation on threonine and tyrosine. A well known substrate for JNK is the transcription factor c-Jun. Phosphorylation by the kinase at the N-terminal serines S63 and S73 leads to its activation (Minden et al. 1994). Transcription factors like ATF-2 and GABP become also activated through JNK (van Dam et al. 1995; Livingstone et al. 1995). Scaffold proteins; also play an important role in mediating the spatial and temporal specificity of the stress cascade activation. Association of JNK with MKK7 and MLK3 is mediated by the scaffold protein JIP-1 (JNK interaction partner 1) (Whitmarsh et al. 1998; Whitmarsh et al. 1998a).

I-1.2.2. p38-MAP kinase cascade p38 was first described as a 38 kDa polypeptide that undergoes Tyr phosphorylation in response to endotoxin treatment and osmotic shock as well as an upstream kinase of MK2 in either IL-1 treated or arsenite stimulated cells (Freshney et al. 1994; Rouse et al. 1994; Han et al. 1994). Until now, four different isoforms of p38 were identified - p38α, p38β, p38γ and p38δ - all of them undergo dual phosphorylation Y as well as T residues between subdomains VII and VIII. p38-alpha is strongly activated in vivo by different environmental stresses like UV light, osmotic and heat shock, by pro-inflammatory cytokines like TNFalpha (tumor-necrosis-factor a) and IL-1 (interleukin-1), and by chemical reagents like anisomycin and arsenite (Kyriakis et al. 1995; Raingeaud et al. 1995). The phosphorylation motif of this MAPK consists of a TEY sequence, which becomes activated through MKK3 and MKK6 (for review see (Ashwell JD 2006)). Although MKK4 can also influence p38 activity, only MKK6 plays the decisive role in this activation (Raingeaud et al. 1996). Activated p38-alpha in turn phosphorylates the MAPKAP kinases 2 and 3 (Freshney et al. 1994; Rouse et al. 1994; Ludwig et al. 1996; McLaughlin et al. 1996) as well as the MEF2 (myocyte enhancer factor) transcription factors (Han et al. 1997). The transcription factor ATF2 is also substrate for p38 (Raingeaud et al. 1995). The domain of the p38 kinase that binds to the substrate and activator is called common docking (CD) domain. This domain 9

varies among MAPKs and even differs in different isoforms of p38. However, the CD domain alone does not determine the docking specificity and another domain named either ED or TT in p38 and ERK2 (Tanoue et al. 2001) is required. Specific binding and phosphorylation of the substrate by a kinase can be prevented by kinase-specific inhibitors. Inhibitors can compete with the ATP for binding near or at the ATP-binding pocket of the catalytic domain thus blocking ATP-binding and phosphorylation of substrate. There are different pyridinyl imidazoles, which were originally identified as a suppressor of cytokine synthesis and later turned out to act as specific inhibitors of p38-alpha and beta (Lee et al 1994; Young et al 1997). Among the different small molecules, SB203580 has been widely used as p38 inhibitor as it inhibit p38α and β specifically. However SB203580 also inhibits cyclo-oxygenases 1 & 2, cytochrome P450s, thromboxane synthetase, TGF-β, lck, c-Raf and some extent JNKs (Borsch-Haubola et al. 1998, Hall Jackson et al. 1999, Eyers et al. 1998, Eyers et al. 1998 (b), Clerks et al. 1998).

I-1.2.3. Physiological roles of JNK and p38 MAPK cascades The JNK and p38 signaling pathways are implicated in several physiological processes spanning from cell differentiation (Adams et al. 2000; Meriane et al. 2000), cytokineproduction (Hoffmeyer et al. 1999; Goebeler et al. 1999), to induction of apoptosis (Kharbanda et al. 1995; Pandey et al. 1996; Kharbanda et al. 2000). p38 kinase was also shown to regulate the activity and thus to control differentiation processes such as myogenesis (Han et al. 1997; Yang et al. 1999; Zhao et al. 1999; Zetser et al. 1999; Wo et al. 2000) and to regulate neuronal differentiation (Okamoto et al. 2000). Apart from this, p38 was shown to take part in cardiovascular development (Yang et al. 2000; Kolodziejczyk et al. 1999), suggesting a crucial role of phosphorylation as a process for regulating the MEF2 transcriptional activity. Recent studies showed that stress- and mitogen-induced kinase cascades are linked together at different points. Often the level of convergence of cascades occurs on the level of transcription factors like ATF-2 (Ruckdeschel et al. 1997). Some kinases like Tpl-2 (Salmeron et al. 1996) or MLK3 (Hartkamp et al. l 1999) are activate more than one downstream kinase. MAPKAP-kinases were also shown to receive signals from different MAP kinases; this includes MAPKAP Kinase-3 (3pK), MNK1/2 (MAPK-interacting kinases 1/2) and MSK1 (mitogen- and stress-activated protein kinase-1), which were activated through ERK and p38 phosphorylation (Ludwig et al. 1996; Aimond et al. 2000; Ryder et al. 2000; Fukunga et al. 1997; Ludwig et al. 1998). 10

p38-alpha gene knock-out causes embryonic lethality because of placental defects (Adams et al. 2000). The mice died at day 11 of gestation. Other suggested that cause of death is failure of erythropoiesis that lack erythropoietin (Epo) gene expression (Tamura et al. 2000). p38beta knock out has no obvious phenotype, and it seems p38-alpha isoform is mainly required for cytokine regulation and T-cell development. (Beardmore et al. 2005). JNK 1 and 2 gene inactivation cause defective T-cell differentiation to Th1 and 2 (Dong et al. 1998, Yang et al. 1998), defective T-cell proliferation, IL-2 production or overproduction, neural tube disclosure (Sabapathy et al. 1999, Kuan et al. 1999 and Dong et al. 2000). JNK3 is reported to display resistance to excitotoxic neural cell death (Yang et al. 1997).

I-1.3. ERK5-kinase cascade ERK5 was first found as a novel MEK5-binding protein and specifically interacts with the dominant negative MEK5 in yeast two-hybrid system (Zhou et al. 1995). ERK5 is a largest member of MAPKs family with primary structure consisting of an 815 amino acid peptide chain. Mouse ERK5 shows 92% amino acid sequence identity with human ERK5. The kinase domain is located in the N-terminal part and is highly conserved including the dual phosphorylation site (T 219 and Y 221) in the TEY motif. The N-terminal domain from amino acids 1-77 is important for cytoplasmic targeting; a domain within amino acids 78-139 is required for association with the upstream kinase MEK5; and another region (aa 140-406) is necessary for oligomerization (Yan et al. 2001). The C-terminus contains a putative NLS (nuclear localization signal) suggesting a role in nuclear translocation of ERK5. ERK5 contains a proline-rich motif possibly targeting ERK5 to the cytoskeleton. Certain prolinerich regions are also known to bind to SH3 (Src homology domain 3) domains. They are found in many proteins involved in tyrosine kinase signaling and cytoskeletal organization (for review see (Wang et al. 2006; Buday et al. 1999). ERK5 exists as constitutive oligomers in unstimulated cells (Yan et al. 2001) while, in contrast, ERK1/2 oligomerize upon phosphorylation. ERK5 over-expression in both stimulated and unstimulated cells leads to oligomerization suggesting that oligomerization does not depend on the phosphorylation status of the kinase. While possessing a TEY motif, and thus resembling the classical ERKs, ERK5 is strongly activated by osmotic, oxidative or fluid shear stress (Abe et al. 1996; Yan et al. 1999). Recent studies reveal that growth factors like EGF (Chao et al. 1999; Kamakura et al. l 1999; Kato et al. 1997) or NGF and serum (Kamakura et al. 1999) are also activators of ERK5. 11

Activation is mediated by diverse stimuli couples the MEK5/ERK5 signaling pathway to transcription factors enhancing their activity. ERK5 has been shown to up-regulate the transcription from several immediate early gene promoters, e.g. the c-jun promoter (Koto et al. 1997; Chyama et al. 2001), or the c-fos SRE (serum responsive element) via Sap1 (Kamakura et al. 1999). Further-more ERK5 was implicated in c-myc phosphorylation (English et al. 1998). Pearson et al. suggested cooperation between MEK5/ERK5 and ERK2 to regulate NF-kB activity (Pearson et al. 2000). The best-characterized targets of ERK5 so far are the members of the MEF2 (myocyte enhancer factor) family.

I-1.4. ERK7/8 signaling pathways The newest member of MAPKs is ERK7 (rat, MAPK15) (Abe et al. 1999) and its homolog ERK8 (human, MAPK15) (Abe et al. 2002). ERK7 is constitutively active and localized in nucleus. ERK7 can also be auto-activated and its C-terminus is important for localization and activation (Abe et al. 1999). It is regulated by proteasomal pathway (Kuo et al. 2004). ERK8 can be activated by serum via protein kinases of the src family and MEK is not needed for its activation (Abe et al. 2002). A recent study shows that ERK8 can down-regulate the transactivation of the glucocorticoid receptor via Hic-5 protein (Saelzler et al. 2006). Both ERK7 and ERK8 can regulate the estrogen receptor degradation (Henrich et al. 2003).

I-1.5. ERK3/4 signaling pathways

I-1.5.1. ERK3/4- identification and structure ERK3 was first identified by Baulton et al. in 1991 together with ERK1/2. A year later in 1992, Gonzalez et al. described ERK4. ERK3 and ERK4 have high homology (62% identity) especially within the predicated kinase domain (73%). ERK3 and ERK4 do not contain the conserved activation loop motif TXY that is present in all other MAPKs. Instead of the TXY motif ERK3/4 display a SEG motif between kinase sub-domains VII and VIII. Even the APE motif of sub-domain VIII, which is extremely conserved in other MAPKs, is replaced by a SPR in ERK3 and ERK4 (cf. Figure 2). A unique feature of ERK3 is its C-terminal domain of about 400 amino acids with no homology to other known proteins, which is only partially present in ERK4 (270aa).

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Figure 2: Sequence alignment of mouse MAPKs. Alignment of the sequences of the catalytic domains of mouse ERK4 (amino acids (aa) 153-218), ERK3 (aa 155-220), ERK7 (aa 152-202), ERK1 (aa 171-235), ERK2 (aa 151-215), ERK5 (aa 186-245), Jun N-terminal kinase (JNK) 1 (aa 155-215) and p38 α (aa 154-212) in the region between sub-domains VII and VIII containing the activation loop (a-loop). Identities are shaded. The presence of an SEG motif in ERK4 instead of TXY that is typical for other MAPKs and SPR in place of APE is marked by asterisks.

I-1.5.2 ERK3 signaling pathways ERK3 is present in both nuclear and cytoplasmic compartment of the cell in contrast to the earlier report that it is a nuclear protein (Cheng et al. 1996). ERK3 expression is ubiquitous in human and mouse. It has been shown that PKCβ can activate ERK3 (Sauma et al. 1996) and an association of ERK3 with B-Raf has also been reported (Kim et al. 1996). The serine residue at position 189 (S189) of conserved activation loop in ERK3 can be phosphorylated by a partially purified and characterized ERK3 kinase (Cheng et al. 1996) and ERK3 itself displays kinase activity against in vitro substrates such as myelin basic protein or histone H1 (Zhu et al. 1994).

S186

ERK4 312

462

583

S189

ERK3

316

481

720

S/T Kinase domain Figure 3 (A): Schematic representation of the primary structure of ERK4 and ERK3. Schematic primary structure of ERK4 and ERK3 showing their N-terminal kinase domain (blue) and long C-terminal where in the beginning they are similar (red) but differ at C-terminus.

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ERK4 ERK3 Prim.cons.

10 20 30 40 50 60 | | | | | | MAEKGDCIASVYGYDLGGRFIDFQPLGFGVNGLVLSATDSRACRKVAVKKIVLSDARSMK MAEKFESLMNIHGFDLGSRYMDLKPLGCGGNGLVFSAVDNDCDKRVAIKKIVLTDPQSVK MAEK DLG R D PLG G NGLV SA D VA KKIVL D S K

ERK4 ERK3 Prim.cons.

70 80 90 100 110 120 | | | | | | HALREIKIRRLDHDNIVKVYEVLGPKGSDLQ---GELFKFSVAYIVQEYMETDLACLLE HALREIKIRRLDHDNIVKVFEILGPSGSQLTDDVGSLTELNSVYIVQEYMETDLANVLE HALREIKIRRLDHDNIVKV E LGP GS L DDVG L YIVQEYMETDLA LE

ERK4 ERK3 Prim.cons.

130 140 150 160 170 180 | | | | | | QGTLTEDHAKLFMYQLLRGLKYIHSANVLHRDLKPANIFISTEDLVLKIGDFGLARIVDQ QGPLLEEHARLFMYQLLRGLKYIHSANVLHRDLKPANLFINTEDLVLKIGDFGLARIMDP QG L E HA LFMYQLLRGLKYIHSANVLHRDLKPAN FI TEDLVLKIGDFGLARI D

ERK4 ERK3 Prim.cons.

190 200 210 220 230 240 | | | | | | HYSHKGYLSEGLVTKWYRSPRLLLSPNNYTKAIDMWAAGCILAEMLTGKMLFAGAHELEQ HYSHKGHLSEGLVTKWYRSPRLLLSPNNYTKAIDMWAAGCIFAEMLTGKTLFAGAHELEQ HYSHKG LSEGLVTKWYRSPRLLLSPNNYTKAIDMWAAGCI AEMLTGK LFAGAHELEQ

ERK4 ERK3 Prim.cons.

250 260 270 280 290 300 | | | | | | MQLILDTIPVVREEDKEELLRVMPSFVS-STWEVKRPLRKLLPDVNSEAIDFLEKILTFN MQLILDSIPVVHEEDRQELLSVIPVYIRNDMTEPHRPLTQLLPGISREALDFLEQILTFS MQLILD IPVV EED ELL V P E RPL LLP EA DFLE ILTF

ERK4 ERK3 Prim.cons.

310 320 330 340 350 360 | | | | | | PMDRLTAEMGLQHPYMSPYSCPEDEPTSQHPFRIEDEIDDIVLMAASQSQLSNWDRYPVS PMDRLTAEEALSHPYMSIYSFPTDEPISSHPFHIEDEVDDILLMDETHSHIYNWERYHDC PMDRLTAE L HPYMS YS P DEP S HPF IEDE DDI LM S NW RY

ERK4 ERK3 Prim.cons.

370 380 390 400 410 420 | | | | | | LSSDLEWRPDRCQDASEVQRDPRAGSTPLAED-VQVDPRKDSQSSSERFLEQ-------QFSEHDWPIHNNFDIDEVQLDPRALSDVTDEEEVQVDPRKYLDGDREKYLEDPAFDTSYS S W D EVQ DPRA S E VQVDPRK E LE PAFDTSYS

ERK4 ERK3 Prim.cons.

430 440 450 460 470 480 | | | | | | -------SHSSMERAFEADYGRSCDYKVGSPSYLDKLLWRDNKPHHYSEPKLILDLSHWK AEPCWQYPDHHENKYCDLECSHTCNYKTRSSPYLDNLVWRESEVNHYYEPKLIIDLSNWK C YK YLD L WR HY EPKLI DLS WK

ERK4 ERK3 Prim.cons.

490 500 510 520 530 540 | | | | | | QAASAPP--------------RAAVAADPVSR---EDE---------------------EQSKEKSDKRGKSKCERNGLVKAQIALEEASQQLAERERGQGFDFDSFIAGTIQLSAQHQ A A S E E

ERK4 ERK3 Prim.cons.

550 560 570 580 590 600 | | | | | | ------------PASLFLEIAQWVKSTQSGSERASPPPDAPEPRLSASPPGHPTPIDGGSADVVDKLNDLNSSVSQLELKSLISKSVSREKQEKGRANLAQLGALYQSSWDSQFVSGGE LE S GGE

ERK4 ERK3 Prim.cons.

610 620 630 640 650 660 | | | | | | ---ASPQFDLDV-----------FISRALKLCTKPED--------LPENKLGD------ECFLISQFCCEVRKDEHAEKENTYTSYLDKFFSRKEDSEMLETEPVEEGKRGERGREAGL QF V S K ED E K G

ERK4 ERK3 Prim.cons.

670 680 690 700 710 720 | | | | | | ----------------------------LNGACISEHPGDLVQTEAFSKERW-------LSGGGEFLLSKQLESIGTPQFHSPVGSPLKSIQATLTPSAMKSSPQIPHKTYSSILKHLN L P

Figure 3(B): Primary structure of mouse ERK3 and ERK4. Sequence alignments of mouse ERK3 and ERK4 and identical amino acid residues are denoted by red color.

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Recently, it became clear that ERK3, unlike other ERKs, is an unstable protein containing two destabilization regions in the N-terminal part of kinase domain, which is constitutively degraded by the proteasome pathway in proliferating cells (Coulombe et al. 2003). During differentiation, ERK3 is stabilized by an unknown mechanism and if the stable ERK3 mutant accumulates in the cell then it causes cell cycle arrest in G1 phase (Coulombe et al. 2003). Interestingly, ERK3 carries a nuclear export signal (NES), which interacts with exportin-1, and nucleo-cytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression (Julien et al. 2003). It has been shown that ERK3 expression is up regulated in 10 out of 21 cancers (Wang et al. 2000). During mouse embryogenesis, ERK3 mRNA shows a sharp peak of expression at embryonic day E11, while only weak expression can be detected at E13 and E15 (Turgeon et al. 2000). Recently, it has been shown that ERK3 can interact with Cyclin D3 and its association with MAP2 is responsible for its involvement in glucose induced insulin secretion (Sun et al. 2006; Anhe et al. 2006).

I-1.5.3 ERK4 signaling pathways Mouse ERK4 shows 93% and 95% sequence identities with human and rat ERK4, respectively. The kinase domain is present at N-terminus of the protein and like ERK3 it possesses an atypical long C-terminus. ERK4 has a serine residue at position 186 (S186) in its activation loop similar to ERK3 (S189). while Erk3 expression is ubiquitous, Erk4 is mainly expressed in human brain, heart and lung and only detectable in brain and heart in rat. Human Erk4 (MAPK4) was mapped on chromosome 18q12-21 (Li et al. 1994) and a cDNA for the rat homolog rMNK2 was isolated (Garcia et al. 1996). Recently, Erk4 (MAPK4) involvement in cardiac dysfunction has been reported as it has been up regulated by 5 fold (Chen et al. 2003). Diethyl maleate treatment also increased Erk4 (MAPK4) expression in HepG2 cells (Casey et al. 2002). Stimuli, activators or relevant substrates of ERK4 have remained elusive and enzymatic activity of the atypical ERKs is not well defined so far.

I-1.6 MAPKAP Kinases (MKs) Three members of MAPKAP kinase (MK) family are known. These are MK2, MK3 and MK5 (for review see (Gaestel 2006)). MK2 was discovered as early as 1992 by Philip Cohen and colleagues as a protein kinase activated by ERK1/2 from rabbit skeletal muscle (Stokoe et al. 1992). MK3 was later discovered in a two hybrid screen with p38 as bait (MacLaughlin 15

et al. 1996; Sithanandam et al. 1996). MK2 homologues have been identified in D. melanogaster and C. elegans and have about 60% amino acid identity with human MK2 (Larochelle et al. 1995). Interestingly, the yeast protein kinases Rck1 and Rck2 show some homology with MK2 (about 35% amino acid identities) and represent the sole MK-related kinases identified to date in the S. cerevisiae genome (Ramne et al. 2000). MK2 has high homology with MK3 (75% amino acid identity) and this discrete them as a subfamily of related kinases. The kinase domains of MK2 and -3 are similar (35 to 40% identity) to CaMK, glycogen phosphorylase b kinase, and the CTKD isoform of RSK. MK2 and 3 kinases can be activated by p38α andβ and this activation could be inhibited by SB203580 (Rouse et al. 1994; MacLaughlin et al. 1996). Mouse MK2 is activated by phosphorylation on T205 and T317 (Engel et al. 1995). The activation leads to the translocation of complex to cytoplasm. Both, MK2 and 3 can phosphorylate Hsp25 in vivo (MacLaughlin et al. 1996), thus playing an important role in stress-induced regulation of Hsp25 chaperone function. It has been also shown that MK2 is able to phosphorylate CREB, SRF, ATF-1 and E47 (Tan et al. l 1996; Sun et al. 1996; Heidenreich et al. 1999; Neufeld et al. 2000). Simultaneously it has been shown by two independent groups that MK2 and its yeast homologues Rck1 and Rck2 play role in the regulation of the cell cycle. While Rck1 and Rck2 were shown to inhibit meiosis in S. cerevisiae, MK2 was proposed to be a check point kinase in mammalian cells (Manke et al. 2005). Another important aspect of MK2-dependent regulation of cellular stress response occurs through the regulation of mRNA stability via AU-rich elements. A good example of such regulation is cytokine production. In fact, MK2-mediated stabilization of AU-rich element of TNF-alpha mRNA leads to inflammatory response (Winzen et al. 1999; Kotlyarov et al. 1999). Recent data about interaction of MK2 and MK3 with the members of polycomb group of proteins leads to novel role of MK2 in chromatin remodeling (Voncken et al. 2005). MK5 (p38-regulated/activated protein kinase) was discovered through homology searches of expressed sequence tag database with the RSK and MK2 sequence as the query (Ni et al. 1998; New et al. 1998). MK5 has 40% amino acid identity with MK2 and MK3, indicating that MK5 is a more distant homologue of these proteins. Indeed, MK5 may have originated earlier during evolution from a common ancestral protein, because MK2 and -3 and MK5 have similar identity with MK2 homologues found in D. melanogaster and C. elegans. Like MK2 and MK3, the kinase domain of MK5 is similar to that of CaMK-related kinases. MK5 possesses NES and NLS overlapping at C-terminus of protein similar to MK2 and 3, another 16

difference is the missing regulatory phosphorylation site C-terminal to the catalytic domain. It is also clear that MK5 shuttles in between nucleus and cytoplasm in exportin-1 dependent manner. However, at the beginning of my work, very little was known about MK5 activation mechanism and its function.

I-1.7 MAP Kinase phosphatase After activation each phosphorylation-regulated protein kinase is needed to be inactivated by dephosphorylation. A specific group of phosphatases, designated as MAP kinase phosphatase (MKPs), is responsible for MAPKs inactivation. Until now, eleven members of this dual specific phosphatase family have been reported. Even though the MKPs share sequence homology with each other, they are specific for substrates. MKPs also differ in their tissues distribution, sub-cellular localization and inducibility by the stimuli. MKP-1 can inactivates all the three MAPKs (ERK1/2, JNK and p38) (Sun et al. 1993), but MKP-3 selectively inactivates ERKs (Muda et al. 1996). MKP-5 and 7 are responsible for inactivation of the p38 pathway (Tanoue et al. 1999 and 2001) and MKP-2 can inactivate JNKs.

17

II. RESULTS

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1. Scaffolding by ERK3 regulates MK5 in development (EMBO J, 2004)

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The EMBO Journal (2004) 23, 4770–4779 www.embojournal.org

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2004 European Molecular Biology Organization | All Rights Reserved 0261-4189/04

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EMBO JOURNAL

Scaffolding by ERK3 regulates MK5 in development Stefanie Schumacher1, Kathrin Laaß1, Shashi Kant1, Yu Shi1, Axel Visel2, Achim D Gruber3, Alexey Kotlyarov1 and Matthias Gaestel1,* 1 Medical School Hannover, Institute of Biochemistry, Hannover, Germany, 2Max Planck Institute of Experimental Endocrinology, Hannover, Germany and 3Department of Pathology, School of Veterinary Medicine Hannover, Hannover, Germany

Extracellular-regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C-terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPKactivated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear-cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C-terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild-type mice is maximum, indicating a role of this signalling module in development. The EMBO Journal (2004) 23, 4770–4779. doi:10.1038/ sj.emboj.7600467; Published online 11 November 2004 Subject Categories: signal transduction; development Keywords: MAP kinases; MAPKAP kinases; nucleocytoplasmic translocation; protein phosphorylation

Introduction Besides the well-known members of the extracellular-regulated mitogen-activated protein kinases (MAPKs), ERK1 and ERK2, which are central members of this MAPK pathway (Boulton et al, 1991; Johnson and Lapadat, 2002), several other ERK-related genes and corresponding proteins were identified such as ERK3 (MAPK6) (Zhu et al, 1994; Meloche et al, 1996), ERK4 (ERK3-related, ERK3b, p63 MAPK, MAPK4) (Gonzalez et al, 1992), ERK5 (BMK) (Zhou et al, 1995), ERK7 (Abe et al, 1999) and ERK8 (Abe et al, 2002). Of *Corresponding author. Medical School Hannover, Institute of Biochemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: þ 49 511 532 2825; Fax: þ 49 511 532 2827; E-mail: [email protected] Received: 19 July 2004; accepted: 11 October 2004; published online: 11 November 2004

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these, only ERK3 and ERK4 lack the characteristic activation loop signature TEY and instead display a SEG motif. The serine residue within this motif (S189 in ERK3) can be phosphorylated by a partially purified and characterised ERK3 kinase (Cheng et al, 1996) and ERK3 itself displays kinase activity against in vitro substrates such as myelin basic protein or histone H1 (Zhu et al, 1994). A unique feature of ERK3 is its C-terminal domain of about 400 amino acids with no homology to other proteins, which is only partially present in ERK4 (170 amino acids). So far, relevant stimuli, activators and in vivo substrates for ERK3 and ERK4 have not been identified. Recently, it became clear that ERK3, unlike other ERKs, is an unstable protein containing two destabilisation regions in the N-terminal kinase lobe, which is constitutively degraded by the proteasome pathway in proliferating cells (Coulombe et al, 2003). During differentiation, ERK3 is stabilised by an unknown mechanism and its intracellular accumulation is paralleled by cell cycle arrest in G1 (Coulombe et al, 2003). Interestingly, ERK3 carries a nuclear export signal (NES), which interacts with exportin 1, and nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression (Julien et al, 2003). During mouse embryogenesis, ERK3 mRNA shows a sharp peak of strong expression at embryonic day (E)11, while only weak expression can be detected at E13 and E15 (Turgeon et al, 2000). Downstream to MAPKs, there exists a family of MAPKactivated protein kinases (MKs; for a recent review, see Roux and Blenis, 2004). Based on their sequence homologies, the MKs can be classified into five subgroups. Besides the RSK, MSK, MNK and MK(MAPKAPK)2/3 subfamilies, the kinase MK5 is regarded the only member of the fifth subgroup (Roux and Blenis, 2004). MK5 displays about 40% amino-acid sequence identity with the p38 MAPK-activated kinases MK2 and MK3 (New et al, 1998; Ni et al, 1998; Underwood et al, 2003). Similar to MK2 and MK3, MK5 carries a nuclear localisation signal (NLS) C-terminal to its kinase domain, which causes nuclear accumulation of the kinase in resting cells (Seternes et al, 2002; New et al, 2003). Besides the regulatory phosphorylation site at the activation loop, MK2 and MK3 possess another regulatory phosphorylation site in the hinge region between the catalytic domain and the C-terminus (Stokoe et al, 1992; Ben-Levy et al, 1995; Engel et al, 1995). Phosphorylation of this site regulates activity of a C-terminal NES and triggers nuclear-cytoplasmic translocation of MK2 and MK3 (Ben-Levy et al, 1998; Engel et al, 1998; Neininger et al, 2001). Since such regulatory phosphorylation site is not present in the C-terminus of MK5, this kind of coupling phosphorylation-dependent regulation of activity and localisation of MK5 is unlikely. Although MK5 was first described as p38-regulated/activated protein kinase (PRAK) (New et al, 1998), recent data challenged this finding, because endogenous MK5 activity is not significantly increased by stimulation of the p38 MAPK cascade (Shi et al, 2003). In addition, MK5 shows only weak interaction and no & 2004 European Molecular Biology Organization

ERK3–MK5 signalling module S Schumacher et al

stabilisation of endogenous p38 MAPK, as MK2 did (Shi et al, 2003). MK5 displays in vitro activity against the small heat shock proteins Hsp25 (mouse) and Hsp27 (human), but in MK5-deficient cells no reduction of Hsp25 phosphorylation in response to stress could be detected, indicating that other protein kinases such as MK2 and MK3 are responsible for stress-induced phosphorylation of these proteins in vivo (Shi et al, 2003). Hence, as for ERK3, stimuli, activators and physiological relevant substrates for MK5 remain to be identified.

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Specific interaction between MK5 and ERK3 Since the mechanism of activation of MK5 is unclear, we were interested in identification of MK5 interacting partners. A two-hybrid screen using two different prey libraries, mouse 11-day-old embryo and adult mouse brain, was applied. Mouse MK5 and the structurally related kinase MK2 (Engel et al, 1993) were used as baits and analysed in more than 107 mating events. The MK2 screen led to the identification of p38 MAPKa as prey in nine out of about 200 positive clones, but none of the 52 positive clones of the MK5 screen overlapped with the positive clones from the MK2 experiment. Interestingly, three independent clones contained ERK3-Gal 4 fusion proteins as interacting molecules for MK5. The specific interaction of MK5 with ERK3 but not with p38 MAPK was confirmed by selection of yeast growth on medium lacking leucine, tryptophan, histidine and adenine. After several days, colony growth at the selection medium was observed only for MK2–p38 and MK5–ERK3 matings. However, after 2 weeks of incubation at 301C, we could also detect colonies for the MK2–ERK3 mating (Figure 1A). To compare semiquantitatively p38 and ERK3 interactions with MK5, we used a luminometric b-galactosidase assay for quantification of yeast two-hybrid interactions (Figure 1B). While p38–MK5 interaction leads to about a two-fold increase in b-galactosidase activity compared with the negative control, ERK3–MK5 interaction is monitored by a more than 10-fold increase in enzyme activity, indicating a significantly higher affinity of ERK3 for MK5. By in vitro pull-down of GST-MK5, GST-MK2 and, as control, GST alone using nickel–agarose bound with recombinant hexahistidine-tagged ERK3 (Figure 1C) as well as by probing interaction in HEK293 cells cotransfected with GST-ERK3 or His-ERK3 and MK2- or MK5- tandem affinity purification constructs (Shi et al, 2003) (Figure 1D), we could further demonstrate specific interaction between MK5 and ERK3. For analysing whether endogenous MK5 interacts with ERK3, we transfected mouse embryonic fibroblasts (MEFs) and, as a negative control, MEFs derived from MK5-deficient mice with a biotinylatable tag-fused ERK3 protein. After expression and in vivo biotinylation, cells were lysed, the ERK3 fusion protein together with proteins bound was purified using streptavidin beads and endogenous MK5 protein could be detected by Western blot (Figure 1E). Finally, we analysed whether endogenous ERK3 can be co-immunoprecipitated from MEF lysates together with endogenous MK5 (Figure 1F). For wild-type (WT) and MK2-deficient cells, ERK3 is detectable in Western blot of the MK5 immunoprecipitate, while in the negative control, MK5-deficient cells, no ERK3 could be detected, indicating a complex of endogenous MK5 and ERK3 in vivo. Interestingly, in MK5-deficient MEFs,

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Figure 1 Detection of specific MK5–ERK3 interaction. (A) pACT2p38 or -ERK3 in strain Y187 (MATa) was mated with pGBKT7-, pGBKT7-MK5- or -MK2- transformed AH109 (MATa) and plated on an SDDAHLT plate (left) and an SDDLT plate (right). The positions of the different matings are schematically shown. (B) Quantification of b-galactosidase activity in yeast two-hybrid system. (C) Pulldown of GST-MK5, GST-MK2 and GST alone from the Escherichia coli lysate using recombinant hexahistidine-tagged ERK3 bound to nickel–agarose. Co-purification of GST or GST fusion proteins is detected by Western blot using anti-GST antibodies. (D) MK5–ERK3 interaction in 293 cells transfected with plasmids coding for expression of two differentially tagged forms of ERK3, GST-ERK3 and HisERK3, and cotransfected with MK2 and MK5 tandem affinity purification constructs (Shi et al, 2003). Coomassie protein stain of tandem affinity-purified proteins demonstrates GST-ERK3 and His-ERK3 as specific binding partners for MK5. (E) Binding of endogenous MK5 to a biotinylated ERK3 fusion protein in MEFs (WT). MK5, which can be identified by its absence in the MK5 knockout cells (/), can be purified bound to the in vivo-biotinylated ERK fusion protein but not to the control where the biotinylated 72-amino-acid peptide (derived from the C-terminus amino acids 524–595) of the Klebsiella pneumoniae oxalacetate decarboxylase was fused to b-galactosidase (Schwarz et al, 1988). A similar experiment was carried out also for WT and MK2-deficient MEFs, but no endogenous MK2 can be detected bound to the biotinylated ERK3 fusion protein (data not shown). (F) Co-immunoprecipitation of endogenous ERK3 with endogenous MK5 from MEFs. MK5 was immunoprecipitated from cell lysates and IP was applied to SDS–PAGE and Western blot using ERK3 antibodies (upper panel). Whole cell lysates were analysed in lower panel. The EMBO Journal

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a significant reduction in ERK3 level could also be detected (see below). Coexpression of ERK3 causes cytoplasmic translocation of nuclear MK5 ERK3 contains a functional NES (Julien et al, 2003), while MK5 carries an NLS and displays nuclear localisation in resting cells (Seternes et al, 2002). To test whether physiologically relevant interaction between MK5 and ERK3 is prevented by different localisation, we coexpressed tagged versions of both proteins and analysed their subcellular localisation in HEK293 cells, which do not express endogenous ERK3 mRNA or protein to a detectable level. GFP-ERK3 localisation is almost exclusively cytoplasmic and not changed by coexpression of MK5 or MK2 (Figure 2A). Remarkably, coexpression of hexahistidine-tagged ERK3 completely changes GFP-MK5 nuclear localisation to cytoplasmic, while localisation of MK2 remains nuclear (Figure 2A). As a control, expression of p38 MAPKa could change GFP-MK2’s localisation to cytoplasmic but leaves MK5 in the nucleus (Figure 2A). Similarly, when yellow fluorescent protein (YFP)-ERK3 and cyan fluorescent protein (CFP)-MK5 or CFP-MK2 were cotransfected, a specific translocation of MK5 and not of MK2 to the cytoplasm in ERK3-expressing cells is detected (Figure 2B). In cells cotransfected with lower amounts of plasmids (Supplementary Figure 1), the unstable ERK3 protein could hardly be detected after 24 h, while MK5

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Figure 2 Coexpression of ERK3 changes subcellular localisation of MK5. (A) Localisation of GFP-tagged ERK3 and MK5 analysed by fluorescence microscopy and characterised quantitatively by the nuclear/cytoplasmic localisation index i (lower left in each image; io1 stands for predominantly cytoplasmic localisation, while i41 indicates nuclear accumulation). Cytoplasmic localisation of GFPERK3 is not significantly changed by coexpression of epitope-tagged versions of MK2 (myc-MK2) or MK5 (HA-MK5) in HEK293 cells. Nuclear localisation of GFP-MK5 but not of GFP-MK2 is changed to cytoplasmic by coexpression of His-tagged ERK3. Flag-tagged p38 MAPK completely changes localisation of GFP-MK2 but only slightly shifts localisation of GFP-MK5 when coexpressed. GFP is equally distributed in the cells. (B) YFP-ERK3 and CFP-MK5 or CFPMK2 were cotransfected and detected in parallel. Nuclei are stained using TO-PRO-3 (Molecular Probes, Invitrogen).

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is still detectable and, as expected for stoichiometric deficiency of ERK3, mainly in the nucleus. Subcellular localisation of both ERK3 and MK5 in HEK293 cells is fusion tag- and cell type-independent, since an HA-tagged version of MK5 shows the same translocation as GFP-MK5 and similar localisation was observed also in HeLa cells (Supplementary Figure 2). While the p38 MAPKa,b inhibitor SB203580 (Lee et al, 1994) used in a 10 mM concentration and the export inhibitor leptomycin B (200 nM) inhibit MK2’s translocation (Engel et al, 1998)), these agents are not able to inhibit ERK3dependent translocation of MK5 (Supplementary Figure 3). Obviously, a specific interaction between MK5 and ERK3 in vivo leads to translocation of MK5 by cytoplasmic anchoring of MK5 by ERK3. Coexpression of ERK3 leads to phosphorylation and activation of MK5 We were then interested in whether ERK3 regulates enzymatic activity of MK5. So far, no stimulus that activates ERK3 has been described and it is assumed that ERK3 activity is mainly regulated by degradation-dependent changes of its level of expression during development (Coulombe et al, 2003). Furthermore, no MK5-specific cellular substrate has been identified so far, while small heat shock protein Hsp25 is a suitable substrate for MK5 in vitro (Shi et al, 2003). We expressed His-ERK3 together with a GFP-tagged MK5 in HEK293 cells and analysed the activity of MK5 by immunoprecipitation (IP) kinase assay using anti-GFP antibodies and Hsp25 as substrate. As controls, we analysed coexpression of Flag-tagged p38 MAPKa and GFP-MK5, and treated the cells with sodium arsenite, a strong activator of the p38 MAPK cascade (Rouse et al, 1994). Coexpression of ERK3 leads to more than 10-fold increase of GFP-MK5 activity, which is not further stimulated by arsenite treatment and could not be inhibited by 10 mM SB203580 (Figure 3A and B). In contrast, there is no significant stimulation of MK5 activity by coexpression of p38 MAPK alone, and only weaker (about threefold) stimulation of this activity as a result of coexpression of p38 MAPK and arsenite stimulation, which is completely SB203580-dependent. There is also no significant arsenite stimulation of MK5 in cells expressing only the endogenous p38 MAPK (control in Figure 3A and B; Shi et al, 2003). Phosphorylation of GFP-MK5 parallels its activity towards Hsp25 and, in cells coexpressing ERK3, also phosphorylation of ERK3. Since overexpression of GFP-MK5 in HEK293 cells could provide nonphysiological results by titrating out other signalling components, we decided to analyse endogenous MK5 activity in MEFs dependent on ERK3 expression. WT and, as a negative control, MK5-deficient MEFs were transfected with His-tagged ERK3 and endogenous MK5 activity was determined by IP kinase assay. In this experiment, MK5 activity can only be detected in WT MEFs transfected with ERK3 (Figure 3C), indicating that ERK3 is able to specifically activate endogenous MK5. Catalytic activity of ERK3 is not required for MK5 translocation and activation An obvious mechanism for MK5 activation could be its regulatory phosphorylation in the activation loop at T182 directly by ERK3. To prove this, we investigated whether catalytic activity of ERK3 is necessary in the signalling module. Two ATP-binding pocket mutants and an activation & 2004 European Molecular Biology Organization

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Figure 3 Expression of ERK3 stimulates activity of MK5. (A, B) Coexpression of MK5 and ERK3 in HEK293 cells. (A) Autoradiograph of MK5 IP kinase assay. GFP-MK5 was precipitated by anti-GFP antibodies from unstimulated cells (C), HEK293 cells stimulated by 150 mM arsenite treatment (A) and from arsenitestimulated cells pretreated for 1 h with 10 mM SB203580 (AS) expressing GFP-MK5 alone (Ctr), or coexpressing Flag-p38 or HisERK3. MK5 kinase activity in the IP was determined by incorporation of phosphate from [g-33P]ATP into the in vitro substrate Hsp25. In the IP, ERK3 and GFP-MK5 were also phosphorylated. (B) Quantification of Hsp25 phosphorylation by phospho-imaging of two independent experiments, each with double determinations. (C) Autoradiograph of IP kinase assay using MK5 antibodies for WT and MK5-deficient (/) MEFs transfected with control plasmid (C) or expressing His-ERK3.

loop catalytic dead mutant of ERK3, ERK3-K49,50R, -K49,50A and -S189A, which were tested to be catalytically inactive in a myelin basic protein in gel kinase assay (not shown), were analysed for their ability to translocate and activate MK5 when coexpressed in HEK293 cells (Figure 4). Unexpectedly, all mutants are able to translocate (Figure 4A and not shown) and activate MK5 (Figure 4B). Coexpression of all mutants leads to significant phosphorylation of MK5 and its in vitro substrate Hsp25. Furthermore, a significant phosphorylation of WT ERK3 and all mutants could be observed, suggesting that ERK3 itself might be a direct substrate for MK5. Since the activation loop mutant ERK3-S189A shows comparable phosphorylation to WT ERK3, the putative phosphorylation site(s) must be distinct from S189, a site that is a target of a previously characterised ERK3 kinase (Cheng et al, 1996). & 2004 European Molecular Biology Organization

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Figure 4 Catalytic activity of ERK3 is not necessary for translocation and activation of MK5. (A) GFP-ERK3 and its catalytic dead mutants, GFP-ERK3-K49,50R and GFP-ERK3-S189A, are mainly in the cytoplasmic compartment of HEK293 cells. When coexpressed as His-tagged proteins, ERK3 and its mutants change MK5 localisation from nuclear (control) to cytoplasmic. (B) Upper panel: Phospho-image of IP kinase assay using anti-GFP antibodies and HEK293 cells transfected with GFP-MK5 (control) and cotransfected with ERK3 and its mutants. The positions of His-ERK3 and its mutants, GFP-MK5 and the MK5 in vitro substrate Hsp25 are indicated. Lower panel: Western blot using anti-ERK3 antibody (Santa Cruz, sc156) for the HEK293 lysates used for the IP kinase assay as expression control for His-ERK3 protein and its mutants. (C) Quantification of MK5 and ERK3 activity by phospho-imaging in two independent experiments, each with double determinations.

Identification of C-terminal regions in ERK3 necessary for MK5 binding, translocation and activation Since enzymatic activity of ERK3 is dispensable for MK5 activation, we were interested in whether domains outside the catalytic region of ERK3 are involved. By stepwise deletion of the C-terminal extension of ERK3 (Figure 5A), regions necessary for MK5 translocation and activation were identified. Deletion of the complete C-terminus of ERK3 (amino acids 301–720, ERK3-DC3), which leaves only the catalytic The EMBO Journal

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domain of ERK3 intact, leads to loss of MK5–ERK3 interaction (Figure 5B) as well as of MK5 translocation and activation (Figure 5C and D). In contrast, the mutant ERK3-DC2 (amino

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acids 358–720 deleted) is sufficient for MK5 binding and translocation but not for its activation. Finally, the mutant ERK3-DC1 (amino acids 472–720 deleted) is able to bind, translocate and activate MK5. For effective activation of MK5, the binding region of ERK3 and the resulting translocation are not sufficient, but the region between amino acids 358 and 472 is needed. As for the kinase dead mutants of ERK3, activation of MK5 is paralleled by phosphorylation of ERK3 and of ERK3-DC1 (Figures 4B and 5D), indicating that after activation MK5 might act as a direct ERK3 kinase. The ERK3-related kinase ERK4 (also designated ERK3b or p63 MAPK) (Gonzalez et al, 1992) shares homology with ERK3 within the catalytic domain and, in part, also within the C-terminal extension (Figure 5A). ERK4 also interacts with MK5 in a pull-down assay (not shown) and translocates MK5 to the cytoplasm (Figure 5C). However, ERK4 cannot activate MK5 (Figure 5D), supporting the notion towards a specific contribution of the region between amino acids 357 and 471 of ERK3, which shows a lower degree of homology to ERK4 (cf. Figure 5A). The C-terminus but not the D-domain in MK5 is necessary for ERK3 binding In the C-terminal region of ERK3 necessary for MK5 binding (amino acids 301–357), two common docking (CD)-like motifs (Tanoue and Nishida, 2003), which could interact with basic D-domains of MAPK substrates such as MKs, can be identified. However, these CD-like motifs show some differences from the CD motifs present in ERK1,2, JNKs and p38 MAPKs. Furthermore, mutation of conserved residues, for example D339, does not prevent interaction with MK5 (not shown). Deletion of the D-domain in MK5 (cf. Figure 6A), which overlaps with the NLS of this kinase (Seternes et al, 2002), from RKRK to GTGT (amino acids 361–364, MK5-GTGT) leads to cytoplasmic localisation of MK5 independent of ERK3 (Figure 6D). Interestingly, this mutation does neither block MK5’s interaction with ERK3 (Figure 6B) nor its ERK3-dependent activation (Figure 6C). In contrast, deletion of the C-terminus of MK5 starting from amino acid 369 (MK5-1-368) leads to a mutant with intact Ddomain, which can no longer bind to ERK3 and which is not

Figure 5 Deletion analysis of ERK3–MK5 interaction and ERK3dependent activation of MK5. (A) Schematic representation of the C-terminal deletions of ERK3 used and of the amino-acid sequence similarity between mouse ERK3 (gi 31560797) and mouse ERK4 (gi 27369906). Dark grey: catalytic domain; light grey: parts of Cterminal extention similar between ERK3 and ERK4; white: similarity gap in C-terminus ERK4; hatched: C-terminal regions of ERK3 and ERK4 without sequence similarity. (B) GST-MK5 pull-down of GFP-tagged ERK3 and its mutants from lysates of transfected HEK293 cells. GFP-ERK3 was detected in pull-down and whole lysate (as control) by Western blot against GFP. (C) Subcellular localisation of GFP-ERK3 deletion mutants and of GFP-ERK4 is mainly cytoplasmic. GFP-MK5 is only translocated by the MK5binding ERK3 mutants DC1 and DC2 and by ERK4, which also binds MK5 (not shown). (D) The C-terminal extension of ERK3 (amino acids 358–471) missing in DC2 and DC3 is necessary for activation of MK5 and for subsequent phosphorylation of ERK3. ERK4, which displays a homology gap (see A) in the region between amino acids 358 and 471, is not able to activate MK5. GFP-MK5 activity was monitored by IP kinase assay as shown in Figure 3A. His-tagged versions of ERK3 and ERK4 were coexpressed with GFP-MK5 or, as a negative control, with GFP-MK2, and activity of MK5 was determined by a-GFP IP kinase assay. & 2004 European Molecular Biology Organization

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T182

A

308 368 473

MK5 Kinase 361RKRK364 D motif

M

K5 -G T

GT K5 -1 -3 68 M K5 -T 18 M 2A K5 -K 51 M K5 E

C M

GF M P K5 M -GT K G M 5-T1 T K5 8 -T 2D 18 M 2A K M 5-1K5 36 8

B

- His-ERK3 - GFP-MK5 GST-ERK3 pull-down - Hsp25 - GFP-MK5 - GFP-MK5 1–368

Lysate

D

GFP-MK5

-GTGT

-1-368

-T182A

-K51E

binding mutant MK5-K51E was not phosphorylated on existing T182, indicating that, in complex with ERK3, MK5 activity is necessary for its own phosphorylation and activation, suggesting ERK3-initiated autophosphorylation of MK5. This idea is supported by the observation that no other proline-directed kinases of the ERK, JNK and p38 MAPK family could be detected in the immunoprecipitated ERK3– MK5 complex by Western blot using pan-ERK, pan-JNK and pan-p38 MAPK antibodies (not shown). It is known that the MK5-related enzyme MK2 is translocated to the cytoplasm as a result of phosphorylation at T317 in the C-terminal hinge region independent of phosphorylation of T205 in the activation loop (Ben-Levy et al, 1998; Engel et al, 1998). Similar to MK2, the activation loop mutant MK5-T182A shows also ERK3-dependent translocation to the cytoplasm (Figure 6D). However, in contrast to MK2, phosphorylation of MK5 as well as phosphorylation of ERK3 is not necessary for MK5’s ERK3-driven translocation at all (cf. Figure 6C and D).

Control

+ His-ERK3

Figure 6 Mutational analysis of MK5 properties necessary for ERK3 binding and its activation. (A) Schematic structure of MK5. (B) GSTERK3 pull-down of GFP-tagged MK5 and its mutants (MK5-GTGT— D-domain mutant; MK5-1-368—C-terminal deletion of amino acids 369–473; GFP-MK5-T182A (activation loop)) from lysates of transfected HEK293 cells. GFP-MK5 was detected in pull-down and whole lysate (as control) by Western blot against GFP. (C) Upper panel: Phospho-image of IP kinase assay as in Figure 3A of cells coexpressing His-ERK3 and GFP-MK5 or -MK5 mutants (including also the kinase-dead ATP-binding pocket mutant MK5-K51E). Lower panel: Expression control for GFP-MK5 and its mutants in the cell lysates used for IP kinase assay. (D) GFP-MK5 and its mutants were transiently cotransfected with His-ERK3 or, as a control, transfected alone, and localisation of the GFP fusion proteins was analysed by fluorescence microscopy as described.

translocated and activated (Figure 6). This indicates that ERK3–MK5 interaction proceeds between the region of amino acids 301–358 in ERK3 and 369–473 in MK5 and is different from the CD interaction of other MAPKs with their MKs. Catalytic activity and phosphorylation of T182 in the activation loop of MK5 are necessary for its activation but not for its translocation to the cytoplasm To decide whether MK5 catalytic activity is necessary for ERK3-regulated activation of MK5, we analysed the GFP fusion protein of the activation loop mutant T182A and of the ATP-binding site mutant K51E (Seternes et al, 2002) in the IP kinase assay (Figure 6C). Although it binds to ERK3 (Figure 6B), GFP-MK5-T182A is not phosphorylated as a result of coexpression of His-ERK3 and, as expected, no kinase activity against the substrates Hsp25 and ERK3 can be detected (Figure 6C). This indicates that ERK3-mediated activation of MK5 depends on phosphorylation of its activation loop at T182 and that both Hsp25 and ERK3 phosphorylations are due to MK5 activity. More interestingly, the ATP& 2004 European Molecular Biology Organization

Coexpression of ERK3 and MK5 in mouse embryogenesis It has been recently shown that ERK3 markedly accumulates during differentiation and increased ERK3 level inhibits proliferation by a G1 arrest blocking S-phase entry (Coulombe et al, 2003; Julien et al, 2003). In mouse embryonic development, ERK3 mRNA expression peaks at day E11, while at days E13 and E15 there is only weak expression and at days E9, E17 and P1 no ERK3 mRNA is detectable (Turgeon et al, 2000). We analysed ERK3 and MK5 expression by in situ hybridisation at E11 and E14.5 (Figure 7). ERK3 mRNA is widely expressed in E11 embryos (Figure 7A), while in E14.5 embryos its expression appears to be reduced and signal is restricted to some tissues including lung and subregions of the brain (arrows in Figure 7B). Similar to ERK3, MK5 mRNA seems to be widely expressed at E11 (Figure 7C), but restricted to low levels and few specific sites at E14.5 (arrows in Figure 7D). Background levels of control hybridisation using sense MK5 riboprobe were higher than in the control experiments for ERK3 (not shown);therefore, further studies will be required to define the specificity and extent of overlaps in MK5 and ERK3 expression in more detail. These results do, however, support the notion towards spatiotemporal coexpression of MK5 and ERK3 during mouse embryogenesis. Reduced ERK3 levels in MK5-deficient cells Recently, we generated MK5-deficient mice and could show that these animals do not exhibit a significant phenotype in the mixed 129  C57/B6 genetic background (Shi et al, 2003). Meanwhile, these mice were backcrossed to the C57/B6 genetic background. We decided to analyse ERK3 expression in embryonic cells derived from these mice. Primary embryonic fibroblasts were derived from E12.5 stage of WT, MK5deficient and, as another control, MK2-deficient animals and subjected to Western blot detection of ERK3 and p38 MAPK (Figure 7E). Since MK2 is a major interaction partner of p38 MAPKa, its absence in MK2-deficient cells leads to reduced levels of p38 MAPKa as seen in the control. Interestingly, in MK5-deficient cells, the relatively high level of expression of ERK3 in WT embryonic fibroblasts is significantly reduced, indicating that endogenous MK5 is a major stabilising interaction partner of endogenous ERK3 in these cells. Conversely, The EMBO Journal

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A

C

B

Table I Embryonic lethality of MK5-deficient mice displayed by genetic under-representation of homozygous MK5 mutant mice after embryonic day 11

D

+/+

+/

/

E11–12 (n ¼ 14)

1.0 (4)

2.0 (8)

0.5 (2)

E13–15 (n ¼ 16) P17 (n ¼ 155)

1.0 (5) 1.0 (42)

1.8 (9) 2.2 (91)

0.4 (2) 0.5 (22)

E11–12 (n ¼ 14)

—

1.0 (9)

0.55 (5)

E13–15 (n ¼ 31)

—

1.0 (21)

0.48 (10)

Intercross MK5+/  MK5+/

E

WT

F

MK5−/− MK2−/−

WT

- ERK3 - Actin p38

G

MK5−/− MK5−/− + GFP-MK5 - ERK3 - Actin

H

I

J

Figure 7 Expression of ERK3 and MK5 in mouse embryos. Spatiotemporal coexpression of ERK3 (A, B) and MK5 mRNA (C, D) detected by in situ hybridisation in embryos of stages E11 (A, C) and E14.5 (B, D). (E) Western blot detection of ERK3 in WT, MK5deficient (MK5/) and MK2-deficient (/) primary MEFs obtained from day 12.5 embryos. As a control, the stripped blot was developed with anti-p38 MAPK and anti-b-actin antibodies. (F) Rescue of ERK3 level in MK5-deficient MEFs by transient transfection of GFP-MK5. As control, ERK3 expression in WT MEFs (WT) was analysed. (G) Dead and autolytic MK5/ embryo at day E13.5 with an overall length of approximately 5 mm (asterisk denotes placenta) when WT littermates were approximately 14.5 mm long (H, WT littermate), suggesting premature death of the MK5/ embryo around day E11.5. All organs of the MK5/ embryos were disintegrated and autolytic at day E13.5 (intestine is shown in I) when compared to the organs of WT littermates (WT intestine shown in J). Bars ¼ 1 mm (G, H) and 50 mm (I, J). Photographs were generated from formalin-fixed, paraffin-embedded tissue sections, stained with haematoxylin and eosin.

ERK3 downregulation by siRNA technique or absence of ERK3 due to deletion by homologous recombination also leads to significantly reduced MK5 activity (Seternes and Keyse, personal communication). In SV40 large T immortalised MK5-deficient embryonic fibroblasts (Shi et al, 2003), a significantly reduced expression of ERK3 is also detected (Figure 7F). Since these cells can be efficiently transfected using standard methods, we ask whether reintroduction of MK5 by expression of GFP-MK5 can rescue the ERK3 level. Overexpression of GFP-MK5 leads to strongly increased ERK3 levels in these cells (Figure 7F), further supporting the ERK3stabilising interaction between both protein kinases. 4776 The EMBO Journal VOL 23 | NO 24 | 2004

MK5+/  MK5/

Day

Values represent the relative ratio of genotype frequency compared to WT and MK5+/, respectively. The total number of embryos and newborn animals analysed for a specific cross in a specific stage, n, and the number of organisms representing the different MK5 genotypes are given in parentheses. Genotyping was carried out by PCR as described previously (Shi et al, 2003).

MK5 deficiency causes embryonic lethality around E11 Interestingly, in the C57/B6 genetic background, MK5-deficient mice showed embryonic lethality with incomplete penetrance. Homozygous mutants were under-represented at least after E12, where major deviation from the expected Mendelian ratios was already observed with only about 50% of the expected number of MK5/ embryos detectable (Table I). In addition, we observed an increased number of MK5-deficient autolytic pups from E13 (Figure 7G–J), which may have resulted from developmental defects at earlier times. It is to be noted that autolytic embryos were 5 mm long, a body length that is usually seen around day E11.5, strongly suggesting that embryonic death must have occurred around day E11.5 where ERK3 mRNA expression in wild mice is maximum (Turgeon et al, 2000). These observations support the notion that ERK3 and MK5 cooperate in regulation of mouse development and differentiation at a stage close to E11.

Discussion Specific molecular interaction between the C-terminus of MK5 and amino acids 301–358 in the C-terminal domain of ERK3 results in translocation of MK5 from the nucleus to the cytoplasmic compartment of the cell. Obviously, ERK3 binding and translocation is not sufficient for MK5 activation, which requires a further C-terminal region between amino acids 358 and 471 of ERK3 as well as MK5 catalytic activity itself. The finding that the C-terminal region of ERK3 but not its catalytic activity is necessary for MK5 activation indicates a scaffolding and translocator function of ERK3 for MK5. Since MK5 catalytic activity is required for its own activation and since ERK3 undergoes MK5-dependent phosphorylation, a scaffolding for MK5 by cytoplasmic ERK3 followed by an ERK3-mediated autophosphorylation and autoactivation of MK5 and subsequent phosphorylation of ERK3 by activated MK5 is supposed (cf. Figure 8). In most aspects, this is different from the well-known mechanisms by which a catalytically active kinase stimulates a downstream kinase via phosphorylation of its regulatory sites. It is known that other catalytically active protein kinases, such as the yeast protein kinase Pbs2p, which binds Hog1 and Ste11p, can also & 2004 European Molecular Biology Organization

ERK3–MK5 signalling module S Schumacher et al

MK5 Nucleus

1 2 P

3

MK5 P

ERK3

P

Cytoplasm

Figure 8 Schematic representation of our model of MK5 activation by ERK3. Increased level of cytoplasmic ERK3 leads to increased cytoplasmic anchoring of MK5, which shuttles between nucleus and cytoplasm (1). Cytoplasmic scaffolding of MK5 by ERK3 facilitates activation of MK5 by autophosphorylation (2) and activated MK5 phosphorylates ERK3 (3).

act as scaffold (Posas and Saito, 1997). In the case of the ERK3/MK5 module, no catalytic activity of the scaffolding ‘kinase’ is required for phosphorylation and activation of the downstream element MK5 although ERK3 is known to display some catalytic activity (Julien et al, 2003). Hence, our finding is more similar to the unfolded protein response receptor Ire1, where a conformational change in the catalytically active kinase domain, triggered by occupancy of its active site with a ligand, stimulates all known downstream functions without the need for catalytic activity (Papa et al, 2003), or to protein kinases carrying both catalytic and pseudokinase domains, such as Janus kinases where the pseudokinase domain is necessary for efficient activation of the enzyme (Saharinen et al, 2000). Recently, activation of the protein kinase LKB1, a gene mutated in Peutz–Jeghers cancer syndrome and involved in polarisation of epithelial cells, by the pseudokinase STRAD has been reported (Baas et al, 2003, 2004). In this case, binding between LKB1 and STRAD also induces nuclearcytoplasmic translocation of LKB1, phosphorylation of both LKB1 and STRAD and activation of LKB1 (Baas et al, 2003). It was shown that LKB1 exhibits STRAD-mediated autophosphorylation and that other components in the complex, such as MO25 (Boudeau et al, 2003), may stimulate this process. ERK3 is a protein kinase containing an N-terminal catalytically active domain. The C-terminal extension between amino acids 442 and 720 displays at least weak homology to a protein kinase catalytic domain, since it has been described as a member of an MAPK cluster in euKaryotic Orthologous Groups (KOG0660, NCBI; Marchler-Bauer et al, 2003), which contains also other catalytic kinase domains. Similar to STRAD, essential conserved kinase subdomains are lacking in the C-terminus of ERK3. By the fact that parts of the C-terminal ‘pseudokinase’ domain of ERK3 are essential for MK5 translocation and activation, the role of STRAD for LKB1 is resembled. Furthermore, our observation that a functional ATP-binding pocket of MK5 is necessary for its activation suggests ERK3-mediated autophosphorylation of MK5 similar to STRAD-mediated autophosphorylation of LKB1. Also, both STRAD and ERK3 translocate the target protein to the cytoplasm and are phosphorylated after activa& 2004 European Molecular Biology Organization

tion of their target. Finally, nuclear-cytoplasmic shuttling of both STRAD and ERK3 has been reported to be necessary for cell cycle arrest (Baas et al, 2003; Julien et al, 2003). Hence, this activation mechanism could be of general importance in growth regulation and development and could assign a new role to other pseudokinases lacking residues essential for catalysis. One may speculate that an activation mechanism that does not require catalytic activity of a phosphorylationregulated activator kinase but only its expression and binding to the target kinase may be well suited for more sustained activation of the target kinase during development and differentiation. Apart from transient phosphorylation-dependent signalling, which often occurs in response to extracellular signals and which can be rapidly reverted by dephosphorylation of the activator kinase, changes in expression and stability of pseudokinase-like translocators and activators may add another regulatory level in the orchestration of signalling. With regard to this idea, one should be aware that in the human kinome, 50 protein kinases were identified that lack residues essential for catalysis and are predicted to act as catalytically inactive scaffolding proteins or pseudokinases (Manning et al, 2002). The reason for incomplete penetrance of the embryonic lethal phenotype of homozygous MK5 mutant mice is still enigmatic. The remaining viable homozygous MK5 mutant mice are smaller after birth (e.g. body mass P17: MK5 þ /: 7.670.26 g; MK5/: 6.770.28 g; Po0.001), but do not display morphological or histological abnormalities when analysed after 3, 6 and 24 weeks. We also inspected the maternal placenta of hemizygous MK5 mutants used for the intercrossing, but could not detect abnormalities that explain the incomplete penetrance of the offspring lethality. Hence, the detailed developmental effect of the ERK3/MK5 signalling module in mouse embryogenesis and the MK5 targets involved remain to be identified and knowing the phenotype of the ERK3 knockout mouse will be certainly helpful. Apart from this, understanding of the detailed molecular mechanism leading to ERK-dependent autoactivation of MK5 within this signalling module and of the role of MK5-dependent ERK3 phosphorylation in its regulation, and identification of additional components of the ERK3/MK5 complex require further investigation.

Materials and methods Yeast two-hybrid screen A pretransformed mouse 11-day embryo MATCHMAKER pACT2cDNA library (Clontech MY4012AH) or a mouse brain library (MY4008AH) in strain Y187 (MATa) was mated with pGBKT7-MK5transformed AH109 (MATa) and plated on 20 SDDHLT plates with 15 mM aminotriazol and on 20 SDDAHLT plates. The plates were incubated for 3–21 days at 301C. For semiquantitative luminometric analysis of protein–protein interactions in yeast, the Galacto-Light plus system (Applied Biosystems) was used. Cloning and site-directed mutagenesis For cloning into pENTR/D-TOPO (Invitrogen), mouse ERK3 cDNA was amplified from the identified two-hybrid clone pACT2-cDNAERK3 by PCR using the primer pair 50 -CCA CAT GGC AGA GAA ATT CGA AAG TCT C-30 (forward) and 50 -TTA GTT CAG ATG TTT CAG AAT GCT GC-30 (reverse). For cloning into pEGFP-C1 and pEYFP-C1, pACT2-cDNA-ERK3 was digested by EcoRI, refilled with Klenow enzyme and redigested by XhoI and inserted into BspEI-cleaved, Klenow-filled and XhoI-cut dephosphorylated vector. Site-directed mutagenesis was performed in pENTR/D-ERK3-WT and pEGFP-C1ERK3-WT using the Quik-change XL Site-directed Mutagenesis Kit The EMBO Journal

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(Stratagene). The recombination reaction between the entry clone and the pDEST26 vector for His-tagged ERK3 expression was achieved with the LR Clonase Kit (Invitrogen). C-terminal deletion mutants of ERK3, ERK3-DC1, -DC2, -DC3, were generated from both pENTR/D-ERK3 and pEGFP-C1-ERK3. Most MK5 constructs were a kind gift from Dr Ole Morten Seternes and are described elsewhere (Seternes et al, 2002). An EcoR1/KpnI MK5-coding fragment of pEGFP-C1-MK5 was ligated into EcoR1/KpnI-cut pECFP-C1 to give pECFP-C1-MK5. Expression of fusion proteins in HEK293 and detection of subcellular localisation of GFP fusions A total of 2.5 106 HEK293 cells were transfected using 500 ml Optimem (Gibco) and Lipofectamine PLUS reagent (Invitrogen). A 0.5 mg portion of expression plasmid for GFP-MK5 and 5 mg portion of expression plasmid for His-tagged ERK3 were used in cotransfection experiments for subcellular localisation studies; otherwise, equimolar amounts of plasmids were used. For subcellular localisation of GFP-tagged protein, the transfected cells were replated in Chambered Coverglass (Labtek, Nunc) and analysed using a Leica DM IRBE microscope with the Leica TCS confocal systems program. Nuclear and cytoplasmic fluorescence intensity was determined using MetaMorph software (Universal Imaging Corporation) and the measure pixel option for at least seven randomly chosen cells (nX7). Subcellular localisation index i was calculated for each cell as i ¼ (InIb)/(IcIb), where In is the nuclear fluorescence intensity, Ib is the background intensity and Ic is the cytoplasmic intensity. Hence io1 stands for predominantly cytoplasmic localisation, while i41 indicates nuclear accumulation. In vitro pull-down assay A total of 5 106 transfected HEK293 cells expressing different GST-tagged forms of MK5 or ERK3 were washed with ice-cold PBS and lysed in 1 ml lysis buffer containing 1% (v/v) Triton X-100, 10% (v/v) glycerine, 150 mM NaCl, 50 mM Hepes pH 7.5, 1,5 mM MgCl2 and 1 mM EGTA for 30 min on ice. After centrifugation (16 000 g, 41C), the supernatant was transferred to a new tube and incubated with 25 ml glutathione–Sepharose 4B beads (Amersham Biosciences) overnight tumbling at 41C. Proteins bound were analysed by Western blot against GFP (anti-GFP B2, Santa Cruz). Alternatively, purified GST-tagged protein was incubated with recombinant hexahistidine-tagged ERK3 bound to nickel–agarose (Qiagen). Binding of GST or GST fusion proteins was detected by Western blot using anti-GST antibodies (B14, Santa Cruz). MK2 and MK5 tandem affinity constructs used are described by Shi et al (2003). Purification and detection of biotin-ERK3 binding proteins from MEFs A total of 7 106 immortalised MEFs (Shi et al, 2003) were transfected with 5 mg pcDNA6/BioEase-ERK3 (generated from pcDNA6/BioEase-DEST (Invitrogen) and pENTR/D-ERK3) together with 1 mg pEGFP-C1. After washing and centrifugation in ice-cold PBS, cells were resuspended in lysis buffer containing 50 mM Tris pH 7.8, 150 mM NaCl, 1% NP-40 and 1 mM PMSF and incubated for 15 min on ice. The cleared lysate was incubated with 30 ml streptavidin–agarose suspension (1:1) tumbling overnight at 41C. After six washes, proteins bound to beads were analysed by Western blot using sheep anti-MK5 antibodies (kind gift from Dr P Cohen, Dundee).

IP kinase assays IP kinase assay was performed as described (Shi et al, 2003) using anti-GFP (B2, Santa Cruz) and 25 ml of 50% Protein G–Sepharose suspension (Amersham Biosciences) or sheep anti-MK5 antibodies and the substrate Hsp25. Radioactivity incorporated into Hsp25 was quantified by phospho-imaging using a Fuji Bas-1500. In situ hybridisation Embryos were dissected from timed-pregnant mice at E11 and E14.5. E11 embryos were fixed in 4% paraformaldehyde for 2 h, transferred through a dilution series into Tris–HCl saline buffer containing 0.5 M sucrose for cryoprotection and embedded in 4% gelatin. E14.5 embryos were embedded in TissueTek medium (Sakura) immediately after dissection. All samples were quickfrozen at 601C and cryosectioned at 25 mm thickness. Templates for riboprobes were generated by PCR using genespecific primers with attached SP6- and T7-RNA polymerase recognition sites (capitals in primer sequences). Primers for MK5 were T7-FW (50 -AAG GTA ATA CGA CTC ACT ATA GGG aga gct att tca cag aat cag cc-30 ) and SP6-RV (50 -AGA GAT TTA GGT GAC ACT ATA Gaa aga gca tcc ctc agg agc ttg cat tcg-30 ), covering nucleotide positions 1027–2013 of GenBank entry NM_010765. Primers for ERK3 were T7-FW (50 -AAG GTA ATA CGA CTC ACT ATA GGG aga ccg aga gaa gta tct aga gg-30 ) and SP6-RV (50 -AGA GAT TTA GGT GAC ACT ATA Gaa gag aaa tgt ctg ctg agg ttt ag-30 ), covering nucleotide positions 1484–2458 of GenBank entry NM_015806. Templates were tested for correct size and absence of by-products by agarose gel electrophoresis and sequenced to confirm their identity with expected sequence. Digoxigenin-labelled antisense and sense riboprobes were generated by standard methods with SP6- and T7-RNA polymerase, respectively. In situ hybridisation on cryosections was performed using an automated liquid handling system essentially as described (Herzig et al, 2001). Miscellaneous ERK3 Western blot was performed using anti-ERK3 (I-15) from Santa Cruz. Pathological inspection of mouse embryos was carried out as described (Shi et al, 2003). Supplementary data Supplementary data are available at The EMBO Journal Online.

Acknowledgements We thank Dr Ole-Morten Seternes (University Tromsoe, Norway) for several MK5 constructs, Dr Maria Schubert for the pECFP-MK2 construct, Dr Sir Philip Cohen (University Dundee, Scotland) for the MK5 antibodies, Tatiana Iakovleva for help with mice breeding and genotyping, Polina Spies and Kornelia Maslo for help with in situ hybridisation and Drs Helmut Holtmann and Michael Kracht for critical reading of the manuscript. We also thank Drs Ole-Morten Seternes and Steve Keyse (Cancer Research, Ninewells Hospital, Dundee, Scotland) for communicating results prior to publication. This work was supported by the Research Training Network Programme of the European Community (HPRN-CT-2002-00255), by the DFG and by the German Ministry of Research (01 KW9965).

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kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J 22: 3062–3072 Baas AF, Kuipers J, van der Wel NN, Batlle E, Koerten HK, Peters PJ, Clevers HC (2004) Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 116: 457–466 Ben-Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ (1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol 8: 1049–1057

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ERK3–MK5 signalling module S Schumacher et al

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2. CHARACTERIZATION OF THE ATYPICAL MAP KINASE ERK4 AND ITS ACTIVATION OF THE MAPK-ACTIVATED PROTEIN KINASE MK5 (The Journal of Biological Chemistry, in press)

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JBC Papers in Press. Published on September 13, 2006 as Manuscript M606693200 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M606693200

CHARACTERIZATION OF THE ATYPICAL MAP KINASE ERK4 AND ITS ACTIVATION OF THE MAPK-ACTIVATED PROTEIN KINASE MK5*

1 Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Shashi Kant, Stefanie Schumacher, Manvendra Kumar Singh+, Andreas Kispert+, Alexey Kotlyarov and Matthias Gaestel From the Institute of Biochemistry and +Institute for Molecular Biology, Medical School Hannover, Hannover 30625, Germany Running Title: ERK4 interacts with and activates MK5 Address correspondence to: Matthias Gaestel, Ph.D., Medical School Hannover, Institute of Biochemistry, CarlNeuberg-Str. 1, D-30625 Hannover, Germany, Tel. +49 511 532 2825; Fax. +49 511 532 2827; E-Mail: [email protected] is found in all other MAPKs, but possess a SEG The extracellular-regulated kinase (ERK) 4 sequence instead (Fig. 1A). Even the APE motif of (MAPK4) and ERK3 (MAPK6) are structurally subdomain VIII, which is extremely conserved in related atypical MAPKs displaying major other MAPKs, is replaced by an SPR motif in ERK3 differences only in the C-terminal extension. and ERK4 (Fig. 1A). ERK4 and ERK3 carry long CERK3 is known as an unstable mostly cytoplasmic terminal extensions (Fig. 1B). Human Erk4 was protein which binds, translocates and activates the mapped on chromosome 18q12-21 (7) and a cDNA MAPK-activated protein kinase (MK) 5. Here we for the rat homolog rMNK2 was isolated (8). Stimuli, have investigated stability and expression of activators or relevant substrates of ERK4 have ERK4 and have analysed its ability to bind, remained elusive and enzymatic activities of the translocate and activate MK5. We show that, in atypical ERKs have not been well defined so far. contrast to ERK3, ERK4 is a stable protein which Initially the MAPK-activated protein kinase binds to endogenous MK5. Interaction of ERK4 MK5 (9,10), also known as PRAK, was described as with MK5 leads to translocation of MK5 to the a member of MAPK-activated protein kinases (MKs) cytoplasm and to its activation by family and a downstream target of p38 (for recent phosphorylation. In transfected HEK293 cells, reviews see (11,12)). Previous data suggested that where overexpressed catalytic-dead ERK3 is able MK5 is not a physiological substrate for p38 in vivo to activate MK5, catalytic activity of ERK4 is (13), because the stimuli which activate the p38 necessary for activation of MK5 indicating that pathway fail to activate MK5, and binding of ERK4 directly phosphorylates MK5. endogenous p38 to MK5 is weaker as interaction of Interestingly, ERK4 dimerizes and/or p38 with other established substrates, such as MK2 or oligomerizes with ERK3, suggesting that overMK3. Interestingly, it has been recently demonstrated expressed inactive ERK3 recruits active that MK5 strongly interacts with and is activated by endogenous ERK4 to MK5 for its activation. ERK3 (14,15). Hence, ERK3 and ERK4 cooperate in activation In this study, we characterize expression, of MK5. stability and protein interaction of the ERK3-related kinase ERK4 and analyse its influence on subcellular localisation and activity of MK5. Mitogen-activated protein kinases (MAPKs) represent a family of evolutionary conserved MATERIAL AND METHODS enzymes with a central role in the well-characterized MAPK signalling cascades. A wide variety of Cloning and site directed mutagenesis - For cloning extracellular stimuli serve as activators of MAPK into pENTR/D-TOPO (Invitrogen), the open reading pathways leading to appropriate responses of cells, frame of mouse Erk4 cDNA was amplified from the such as proliferation, differentiation, growth and cDNA clone BC062911 (Open biosystems) by PCR migration. MAPK pathways generally have a threeusing the primer pair 5`-CAC CAT GGC TGA GAA kinase module architecture by which the signal is AGG TGA CTG-3` (forward) and 5`-TCA CCA CCT transmitted from an upstream kinase to a downstream TTC TTT GGA GA-3` (reverse). For cloning into kinase by sequential phosphorylation. MAPKs pEGFP-C1, an ERK4 cDNA fragment was digested comprise four well defined groups (ERK1/2 (1,2), with EcoRI and BamHI after amplification by PCR JNKs, p38s and ERK5 (BMK) (3)) but additional using the primer pair 5`-CTG AGA ATT CAA TGG members including ERK3 (1,4), ERK4 (p63mapk, CTG AGA AAG GTG ACT GC-3` (forward) and 5`ERK3-related, ERK3β, MAPK4, Prkm4) (5) and CCC CTG GAT CCC TCA CCA CCT TTC TTT ERK8 (6) have been identified. GGA G-3` (reverse). The recombination reaction ERK4 (p63mapk) was described in humans between the entry clone and the pDEST15, (5), soon after ERK1, ERK2 and ERK3 were pDEST17, pDEST26, pDEST27 and identified (1). Among MAPKs, ERK4 is most closely pcDNA6/BioEase-DEST vectors for GST-, His- and related to ERK3 displaying 62 % overall amino acid BE-tagged ERK4 expression in bacteria and in sequence identity and 73 % within the predicted mammalian system respectively were achieved with kinase domain. Both kinases do not contain the the LR Clonase Kit (Invitrogen). C-terminal deletion highly conserved activation loop (“a-loop”) motif mutants of ERK4 (Fig. 1B), ERK4∆C1, -∆C2, -∆C3, TXY between kinase subdomains VII and VIII that

and GST pull down was performed by using 25 µl of Glutathione Sepharose beads. Western blot was developed with anti-MK5 antibody (a kind gift from Dr. Sir Philip Cohen). Immunodetection of endogenous MK5/ERK4 complexes – 5 x 106 WT, MK5-/- or ERK3-/- MEF cells were grown in culture and lysed with kinase lysis buffer for 30 minutes on ice. IP of endogenous MK5 was performed by incubation of the 1.5 mg protein lysate (150 µl) with 4 µl anti-MK5 antibody overnight followed by incubation with 15 µl Protein G-Sepharose (Amersham Biosciences) for 1h at 4°. After five washings of the Sepharose beads with IPbuffer, the beads were resuspended and boiled in SDS loading buffer. Western blot was developed using anti-ERK4 antibody (a kind gift from Dr. Ole Morten Seternes). Western blot - The protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, USA). To equalize amounts of protein 4 x Laemmli's sodium dodecyl sulfate (SDS) sample buffer was added (40 % glycerol, 4 % SDS, 4 % βmercaptoethanol, 0.4 M Tris-HCl pH 6.7 and 2 mg/ml bromphenol blue). Samples were boiled and centrifuged. Soluble protein extract was subjected to 10 % polyacrylamide SDS gels and transferred to Hybond ECL membranes (Amersham Biosciences). Blots were incubated for 1 h in PBS-0.1 % Tween 20 (PBS/T) containing 5 % powdered skim milk. After three washes with PBS/T, membranes were incubated for 16 h with the primary antibody at 4°C or 1 h at room temperature and for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Antigen-antibody complexes were detected with an ECL detection kit (Santa Cruz Biotechnology) and the digital chemiluminescence’s images were taken by a Luminescent Image Analyzer LAS-3000 (Fuji film). In vitro pull down and IP-kinase assays - Kinase assays were performed after pull down as described above and elsewhere (13) using 15 µl 50 % Glutathione or Protein G Sepharose suspension (Amersham Biosciences), 2.5 µl of buffer (500 mM Na β-glycerophosphate, 1 mM EDTA at pH 7.4), 10 µg of substrate recombinant Hsp25 in a final volume of 20 µl. Then 5 µl of hot ATP mixture (20 mM MgCl2, 0.5 mM ATP, 0.1 µl of [γ-33P]ATP) was added and the reaction mix was incubated for 10 min at 30°C. Radioactivity incorporated into Hsp25 was quantified by phospho-imaging using a Fuji Bas-1500 and TINA 2.09 software. Expression of fusion proteins in HEK293 and detection of subcellular localization of GFP-tagged proteins – 5 x 106 HEK293 cells were transiently transfected by Lipofectamine in accordance to the manufacture’s protocol (Invitrogen). An equimolar amount was used for each vector. For analysis of subcellular localization, GFP and CFP expression vectors were transfected, cells were re-plated in Chambered Cover-glass (Labtek, Nunc) and analysed using a Leica DM IRBE microscope with the Leica

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were generated from both pENTR/D-ERK4 and pEGFP-C1-ERK4 by using the forward primer as mentioned above for full length ERK4 and different reverse primers: ERK4∆C1 - 5´-CTT GCG GAT CCA TCC TCA CGA GAC ACA GGG T- 3´; ERK4∆C2 - 5´-TCT GGG ATC CTC ACA TCA GCA CGA TGT CGT CGA-3´; ERK4∆C3 - 5´ -TGC GGG ATC CTC ATT GTG AGG TGG GCT CAT CCT- 3´. Site-directed mutagenesis was performed in pEGFPC1 ERK4-WT using the Quick-change XL Sitedirected Mutagenesis Kit (Stratagene). For construction of His-tagged ERK4-K49A,K50A expression vector pEGFP-C1-ERK4-K49A,K50A was cloned into pENTR/D-ERK4 vector as BstXI fragment. Recombination reaction was performed between entry and pDEST26 using LR Clonase Kit. ERK3 constructs were described elsewhere (14). Most MK5-constructs were a kind gift from Dr. Ole Morten Seternes and described elsewhere (16). In vitro pull down – Hexahistidine-tagged ERK4 was expressed in bacteria or the protein was isolated from different tissues of mouse, 1 mg of bacterial or tissue lysate protein was incubated with either 0.1 nmol of recombinant GST, GST-MK2 or GST-MK5 bound to Glutathione Sepharose 4B (Amersham Biosciences). After five washes with IP-Buffer (1 x PBS, 50 mM NaF, 1% TritonX-100, 1 mM Na3VO4) the beads were resuspended in 4 x Laemmli buffer and proteins from the beads were separated by SDS-PAGE, used for Western blotting and developed with anti-His antibody (Penta-His, Qiagen) or anti-ERK3 (D23, Santa Cruz) antibody which cross reacts with ERK4. GST-Pull down from lysate of transfected HEK293 cells - A total of 5 x 106 transfected HEK293 cells expressing GST-tagged forms of MK2, MK5, ERK3 or ERK4 together with differently tagged forms of wild type ERK3, ERK4, MK5 and their mutants were washed with ice-cold PBS and lysed in 1ml lysis buffer containing 1 % (v/v) Triton X-100, 10 % (v/v) glycerine, 150 mM NaCl, 50 mM Hepes pH 7.5, 1,5 mM MgCl2 and 1 mM EGTA for 30 min on ice. After centrifugation (16 000 g, 4°C), the supernatant was transferred to a new tube and incubated with 25 µl Glutathione Sepharose 4B beads (Amersham Biosciences) overnight tumbling at 4°C. Protein interactions were analysed by Western blot using GFP- (anti-GFP B2, Santa Cruz), BE- (StreptavidinHRP, Invitrogen), His- (Penta-His, Qiagen) and GSTantibodies (B14, Santa Cruz). BioEase-pull down - For BE pull down HEK293 cells were co-transfected either with pcDNA6/BioEaseERK4 or -ERK3 and pDEST27-ERK3 or -ERK4 vectors, after 16 h cells were lysed and supernatant applied to pull down with 25 µl Streptavidin agarose beads (Invitrogen). Western blot was developed with anti-GST antibody. GST-ERK4 pull down of endogenous MK5 – 5 x 106 WT and MK5-/- mouse embryonic fibroblast (MEF) cells were transfected with either GST or GST-ERK4 expressing vector. After 16 hours cells were lysed

proteins was independent of the respective tag (Fig. 2). ERK4 interacts with MK5 in vitro as well as in transfected HEK293 cells ERK3 and ERK4 are atypical MAP kinases that possess a long C-terminus and lack the conserved TXY motif. Since Western blot and in situ hybridization analysis detected co-expression of ERK4 and ERK3 with MK5 in mouse (see supplementary Figure), we were interested to study whether MK5, a known partner for ERK3 (14,15), also interacts with ERK4. Recombinant His-ERK4 was expressed in bacteria and in vitro pull down was performed with purified GST-MK5 and GST-MK2 protein. His-ERK4 was found to interact with GSTMK5 as well as with GST-MK2 (Fig. 3A). To further study this interaction in a cellular model, GFP-ERK4 was co-transfected with GST-MK5 and GST-MK2, respectively, in HEK293 cells. In GST pull down, GFP-ERK4 was precipitated with GST-MK5 (Fig. 3B). GFP-ERK3, which binds to MK5, was used as a positive control (Fig. 3B). Only weak interaction of GFP-ERK4 was observed with GST-MK2 (Fig. 3B). Although MK5 shows significant homology to MK2 and both kinases can bind to ERK4 in vitro, the data from co-transfected HEK 293 cells imply that a more specific interaction of ERK4 exists with MK5 in vivo. This notion is supported by the co-localisation studies below. Next, we studied interaction of the endogenous proteins. By using a GST-ERK4 pull down assay, we demonstrated that endogenous MK5 interacts with the overexpressed bait in MEF cells (Fig. 3C). As negative controls, we failed to detect interaction with GST alone in WT cells and with GST-ERK4 in MK5-deficient cells. We then analysed interaction of endogenous proteins in mouse embryonic fibroblasts of different genotypes (WT, MK5-/-, ERK3-/-) using immuno-precipitation with MK5 antibodies and subsequent Western blot-detection of ERK4 in the precipitate (Fig. 3D). ERK4 was co-immunoprecipitated with MK5 in WT and ERK3-deficient cells, indicating an ERK3-independent interaction between both proteins. The specificity of the coimmunoprecipitation was confirmed by the failure to precipitate ERK4 from MK5-deficient cells. To check whether the C-terminal region of ERK4, which does not show significant sequence homology to ERK3, or the region homologous to the MK5 binding site in ERK3 between amino acids 330-340 of ERK3 is important for specific interaction with MK5, we co-expressed GFP-tagged C-terminal deletion mutants (GFP-ERK4∆C1-3, cf. Fig. 1B) together with GST-MK5 in HEK293 cells and performed GST pull down. The result demonstrates that GFP-ERK4∆C1 and 2 interact with GST-MK5 while GFP-ERK4∆C3, which lacks the region between amino acids 326-340, fails to bind (Fig. 3E). Hence, the region in ERK4 homologous to amino acids 330-340 in ERK3, which is essential for ERK3

RESULTS ERK4 is a stable protein Recent data showed that human ERK3 is a highly unstable protein and that its degradation depends on N-terminal ubiquitination and subsequent proteosomal degradation (17). To analyse whether ERK4 shows a similar tendency to rapid proteosomal degradation, mouse cDNAs of GFP-ERK3 and GFPERK4 were expressed in HEK 293 cells and proteins were compared for stability. After 24 hours, protein biosynthesis was inhibited by blocking translation using cycloheximide treatment for 2 and 4 hours. In parallel, the proteasome inhibitor MG132 was applied together with cycloheximide to identify proteasome-dependent degradation of the protein kinases. As previously described for human ERK3 (17), in our experimental system N-terminal GFPtagged mouse ERK3 is an unstable protein with a half life of about 1 hour that undergoes proteasomedependent degradation (Fig. 2). In contrast, there was no significant difference in the protein level of GFPERK4 after 4-hour cycloheximide treatment and no effect of MG132 was detected. This indicates that ERK3- and ERK4-protein stability are differentially regulated. To ensure that protein stability is not altered by the large N-terminal fusion, as described for C-terminal fusions of ERK3 (21), we also investigated stability of ERK4 and ERK3 using the small N-terminal BioEase-tag. The stability of both

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TCS confocal systems program or Visitron Systems and SPOT Advanced Programm. Staining of nuclei was performed by adding TO-PRO3 (Molecular Probes) to mounting medium (1:1000). Calf intestinal alkaline phosphatase treatment – pEGFP-C1-ERK4 plasmid was transfected in HEK293 cells. After 16 hours of transfection cells were lysed in kinase lysis buffer (20 mM Trisacetate, pH 7.0, 0.1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM β-glycerophosphate, 50 mM NaF, 5 mM pyrophosphate, 1 % Triton X-100, 1 mM benzamidine, 0.1 % β-mercaptoethanol, 0.27 M sucrose, and 0.2 mM phenylmethylsulfonyl fluoride). Calf intestinal alkaline phosphatase (CIP) (NEB Biolabs) was used for dephosphorylation. 100 µg lysate protein in 1X NEB buffer 3 were incubated with 10 units CIP at 37°C for different time points. Control lysate was incubated under the same condition but without CIP. Determination of protein stability - HEK293 cells were transfected with GFP-ERK3 and GFP-ERK4 or BE-ERK3 and BE-ERK4 plasmids. After 16 hours cells were treated with cycloheximide (100 µg/ml, Calbiochem) for indicated time points with or without MG132 (20 µM, Biomol). Cells were lysed in kinase lysis buffer, centrifuged and equal amount of lysate was loaded onto a SDS gel. Protein amounts were analyzed by Western blotting against GFP or BE.

ERK4 translocates MK5 from the nucleus to the cytoplasm We next analysed the consequence of ERK4-MK5 interaction for the subcellular localization of both proteins using confocal fluorescence microscopy after transfection of ERK4 and/or MK5 expression plasmids into HEK293 cells. Nuclear counter-stain was performed with the dye TO-PRO. Overexpressed MK5 alone showed predominantly nuclear localization as expected by the presence of a nuclear localization signal (NLS) (16). In contrast, GFPERK4 was mainly present in the cytoplasmic compartment of HEK293 cells (Fig. 4, left panel), similar to ERK3 (14) which carries a functional nuclear export signal (NES) in a region which is homologous to ERK4 (18). Cytoplasmic location of ERK4 results from CRM1-dependent nuclear export since treatment with Leptomycin B (LMB, 10 ng/ml, Sigma) for 8 hours caused equal distribution of GFPERK4 between the nuclear and cytoplasmic compartments of HEK293 cells (not shown). To test the physiological relevance of the interaction between ERK4 and MK5, GFP-MK5, or GFP-MK2 as a control, were co-expressed with GST-ERK4 or His-ERK4 in HEK293 cells. MK5, but not MK2, was almost completely translocated to the cytoplasmic compartment in the presence of both GST- or Histagged-ERK4 as well as in the presence of the ERK3 as positive control (Fig. 4, right panel). In contrast, MK2 was mainly present in the nucleus of HEK293 cells and was not affected in its localization by coexpression of neither GST- or His-ERK4 nor ERK3. Hence, ERK4 specifically translocates MK5 into the cytoplasm. This also supports the notion that ERK4binding is independent of the ERK4-fusion tag and specific for MK5 in vivo.

MK5 phosphorylates ERK4 When ERK4 and MK5 were co-transfected in HEK293 cells, additional low-mobility bands of GFP-ERK4 were observed in SDS-PAGE of cell lysates (Fig. 3B, E). Furthermore, phosphate incorporation was found in bands corresponding to ERK4 or ERK4∆C1 in in vitro kinase assay (Fig. 5A, C) suggesting phosphorylation of ERK4 by MK5. To test this hypothesis, GFP-ERK4 was co-expressed with GST-MK5 in HEK293 cells and subsequently treated with calf intestinal alkaline phosphatase (CIP). After 10 minutes of treatment the lower migrating bands began to disappear (Fig. 6A) indicating that they were due to phosphorylation of ERK4. To determine whether MK5 can directly phosphorylate ERK4, HEK 293 cells were cotransfected with wild-type GFP-MK5 and the kinase dead mutant GFP-MK5-K51E as a control. The additional low-mobility band of ERK4 was observed in the presence of wild-type MK5 but not in the control (Fig. 6B). Thus, MK5 catalytic activity is necessary for phosphorylation of ERK4 that probably occurs in a direct manner. In a further experiment, we

ERK4, but not the ATP-binding site mutant ERK4K49A,K50A, activates MK5 in HEK293 cells ERK3 activates MK5 in HEK293 cells by binding and translocation independent of ERK3 catalytic activity (14). We were interested whether ERK4 regulates MK5 kinase activity in a similar manner. Protein kinases were expressed in HEK293 cells and an in vitro kinase assay was performed after pull down of GST-MK5 using the known in vitro substrate Hsp25. We detected Hsp25 phosphorylating kinase activity and phosphorylated MK5, but also phosphorylated ERK4, and in controls phosphorylated ERK3. This indicates the existence of a productive complex between MK5 and ERK4 and ERK3, respectively. Both co-expressed GFP-ERK4 as well as GFP-ERK4∆C1 activated MK5 (Fig. 5A) and were subsequently also phosphorylated by MK5

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(see below). The activation of MK5 by ERK4 was comparable to the activation by ERK3 and its Cterminal deletion mutants ERK3∆C1, ERK3∆C2. Only ERK3∆C3, which is no longer able to bind and to translocate MK5 into the cytoplasm (14), failed to activate MK5 in this assay (Fig. 5A). To see whether endogenous ERK4 is able to active MK5 in vitro, we immunoprecipitated ERK4 from MK5-deficient MEFs and assayed the immunoprecipitate (IP) in a coupled kinase assay using recombinant MK5 and Hsp25. The IP of ERK4specific antibodies but not of a pre-immune serum (control) was able to phosphorylate and activate MK5 (Fig. 5B). We wondered whether activation of MK5 by ERK4 depends on the catalytic activity of ERK4. Hence, we generated an ATP-binding deficient mutant of ERK4 by site-directed mutagenesis. We replaced the lysine residues at position 49 and 50 of the ATP-binding pocket of ERK4 with alanine residues (K49A,K50A or “KK/AA”). When co-transfected, this kinase ATPbinding mutant was unable to activate GFP-MK5 (Fig. 5C). In the same assay we used the appropriate ERK3 ATP-binding mutant ERK3-K49A,K50A. In contrast to ERK4 and in agreement with previously published data (14), in this experimental system the ERK3 inactive mutant activated MK5 to a certain extent (Fig. 5C), probably by an indirect mechanism (see below). We next studied the effect of ERK4 kinase activity for the cytoplasmic translocation of MK5. The GFPtagged ATP-binding mutant of ERK4-KK/AA was expressed in HEK293 cells. Similar to wild type ERK4 this mutant is localised in the cytoplasm and translocated CFP-MK5 from the nucleus to the cytoplasm (Fig. 5D). Hence, translocation of MK5 to the cytoplasm alone is not sufficient to activate MK5.

to bind MK5 (15), is also important for MK5-binding of ERK4. The interaction between GFP-ERK4∆C1 and 2 and GST-MK5 was almost quantitative and GFP-ERK4∆C1 and 2 could even be detected as Ponceau stained protein band on the filter (not shown).

ERK4 binds to the C-terminus of MK5 To determine the ERK4 binding site on MK5, we used the C-terminal deletion mutants MK5-1-358 – which lacks the p38-binding NLS (amino acids 361364) – and MK5-1-368, which contains the NLS but lacks the further C-terminus (Fig. 7A). After cotransfection of HEK293 cells with GST-ERK4 and GFP-MK5 and the C-terminal deletion mutants, respectively, and GST pull down, interaction was detected by Western blot using GFP antibody. GFPtagged MK3, a kinase which is specifically activated by p38 but not by ERK4, was used as a negative control. Both C-terminal deletion mutants of MK5, MK5-1-368 (Fig. 7B) and MK5-1-358 (not shown), did not bind to ERK4 in this assay. Additionally, ERK4 failed to translocate MK5-1-368 (Fig. 7C) confirming that ERK4 binds, similar to ERK3 (14), to the more C-terminal region of MK5. ERK4 can form protein complexes with itself and with ERK3 Recently, a high throughput study on protein interaction in yeast revealed multimerization of ERK3 (19). To examine whether ERK4 forms homodimers and hetero-dimers with ERK3, HEK293 cells were co-transfected with GST-ERK4 and GSTERK3, respectively, and BioEase (BE)-ERK4 plasmids. Then BE pull-down was performed and analysed by Western blot against GST. Specific binding of GST-ERK4 and GST-ERK3 to BE-ERK4 was observed (Fig. 8A) reflecting the ability of ERK4 to form protein complexes consisting of more than one ERK4 and/or additional ERK3 molecules. A BEpull down experiment with BE-ERK3 gave similar results (Fig. 8B). Therefore, ERK3 and ERK4 are likely to exist in multimeric protein complexes. Since BE-ERK3 pull down also precipitates the C-terminal deletion mutant ERK4-∆C3 (not shown), MK5binding and ERK3-binding regions in ERK4 are different. DISCUSSION Here we have shown that the two atypical ERKs, ERK4 and ERK3, behave similarly with respect to MK5 binding, regulation of its subcellular localization and catalytic activity. Furthermore, ERK4 and ERK3 phosphorylation both depend on the catalytic activity of MK5, probably being direct substrates for MK5. Both atypical ERKs show similar expression patterns during mouse embryogenesis

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with elevated transcript levels in brain, lung and kidney suggesting cooperativity of ERK3 and ERK4 in MK5-activation during development (see Supplementary material). Both proteins are subject to exportin1/CRM1-dependent nuclear export and carry a conserved region C-terminal to the catalytic domain which interacts with a sequence C-terminal to the NLS and p38-binding site of MK5. The region between amino acids 326-340 in ERK4, which is homologous the MK5-binding region of ERK3, is also necessary for MK5 binding. Both ERK4 and ERK3 bind to the region of MK5 C-terminal to amino acid 368, probably between amino acids 423472 (15). As a result of interaction with ERK4 and ERK3, nuclear MK5 is exported to the cytoplasm. After activation of MK5, both ERK3 and ERK4 are phosphorylated depending on MK5 catalytic activity. Furthermore, when co-expressed in HEK293 cells, both ERK3 and ERK4 can be detected in an oligomeric protein complex together with MK5. Binding between ERK4 and ERK3 does neither depend on the C-terminal extension of ERK4 nor on the MK5 binding site indicating direct interaction of the catalytic domains. Despite the many similarities, ERK3 and ERK4 seem to differ at least in two points: First, ERK4 protein is significantly more stable than ERK3 that is rapidly degradated in a proteasome-dependent manner (17). The differences in protein stability are independent of the size of the N-terminal tag, challenging the finding that ERK3 is degraded after ubiquitinylation of the free N-terminus (20) while strengthening the notion that the C-terminal part of ERK3 is also involved in targeting degradation (21). Oligomerization of ERK3 and ERK4 may affect ERK3 and/or ERK4 stability under certain conditions. However, our preliminary experiments analyzing the effect of coexpression of ERK4 on protein stability of ERK3 in HEK293 cells (Kant and Gaestel, unpublished) did not reveal significant differences so far. Second, catalytically-dead ATP-pocket mutants of ERK4 and ERK3 differ in their ability to activate MK5. Upon co-transfection in HEK293 cells, ATPpocket mutants of ERK3 still activated MK5 (14) while ERK4 mutants failed to do so. The ability of ERK3 mutants to activate MK5 obviously depends on the experimental system applied, since both in an in vitro phosphorylation assay with recombinant proteins expressed in insect Sf-9 cells and in transfected HeLa cells MK5 activation depends on catalytic activity of ERK3 (15). Taking into account that ERK3 and ERK4 co-exist in protein complexes in transfected cells, MK5 activation by catalytically dead ERK3 may be explained by its ability to recruit active endogenous ERK4 of HEK293 cells to the MK5-containing complex. Since ERK4 is more stable than ERK3, the steady state level of endogenous ERK4 in HEK293 cells is probably higher than that of ERK3. This would also explain why the kinase-dead ERK4 mutant fails to recruit sufficient ERK3 in HEK293 cells to activate MK5. In addition, because of its molar excess, over-expressed

tested whether catalytic activity of ERK4 is required for MK5 dependent phosphorylation of ERK4. GSTMK5 was co-transfected with GFP-ERK4 or the kinase-dead mutant GFP-ERK4-KK/AA, followed by Western blot against GFP. GFP-ERK4 but not the mutant GFP-ERK4-KK/AA was phosphorylated under these conditions (Fig. 6C). Since ERK4KK/AA is unable to activate MK5 (Fig. 5C), it is likely that phosphorylation of MK5 is a prerequisite for phosphorylation of ERK4 by MK5.

kinase-dead ERK4 can also not quantitatively recruit active endogenous ERK4. Hence, the seemingly different behaviour of ERK3 and ERK4 mutants in activation of MK5 may finally be caused by complex formation and differential stability of ERK3 and ERK4 proteins. Hence, protein stability remains the only clear difference detected for these atypical kinases so far. At the end, we should mention that the physiological role of the ERK4/MK5 signalling module is still enigmatic, since extracellular agonists of MK5 have

not been identified amongst the diverse stimuli analysed at the cellular level (Ref. 13 and Schumacher and Kotlyarov, unpublished). Similar to the ERK3/MK5 module (14), the ERK4/MK5 module may play a more prominent role in embryonic or post-natal development. Compound mouse mutants of the different components of these modules will ultimately provide more detailed insight into the physiological role of these atypical ERKs.

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Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M., Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S. L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C., Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S., Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P., and Vidal, M. (2005) Nature. 437, 1173-1178. Epub 2005 Sep 1128.

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1.

FOOTNOTES *We thank Dr. Ole-Morten Seternes (Tromsoe) for MK5 constructs, ERK4-antibodies and sharing unpublished results, Dr. Sylvain Meloche (Montreal) for ERK3-deficient mouse fibroblasts, Dr. Sir Philip Cohen (Dundee) for MK5-antibodies and Kathrin Laaß (Hannover) for help with the kinase assays. This work was supported by European Community grant RTN-HPRN-CT-2002-00255 and by the Deutsche Forschungsgemeinschaft. We also would like to express our thanks to the MD/PhD program “Molecular Medicine” of the Medical School Hannover. The abbreviations used are: aa, amino acids; BE, BioEase; CFP, cyan fluorescent protein; ERK, extracellularregulated kinase; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; MAPK, Mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; MK, MAPK-activated protein kinase.

Figure 1: Sequence alignment of mouse MAPKs and schematic representation of the primary structure of ERK4 and ERK3 and their deletion mutants. A) Alignment of the sequences of the catalytic domains of mouse ERK4 (amino acids (aa) 153-218), ERK3 (aa 155-220), ERK1 (aa 171-235), ERK2 (aa 151-215), Jun Nterminal kinase (JNK) 1 (aa 155-215) and p38 MAPKα (aa 154-212) in the region between subdomains VII and VIII containing the activation loop (a-loop). Identities are shaded. The presence of an SEG motif in ERK4 instead of TXY that is typical for other MAPKs and SPR in place of APE is marked by asterisks. B) Schematic primary structure of ERK4, ERK3 and the C-terminal deletion mutants used in this study. The position of the serine residue of the SEG-motif and the length of the proteins and their domains are shown. Figure 2: Comparison of protein stability of ERK4 and ERK3. HEK293 cells were transfected with GFPtagged (upper part) and BioEase(BE)-tagged (lower part) ERK3 and ERK4. After 16 hours of transfection, cells were treated with cycloheximide (CHX) to block protein translation for 2 and 4 h and for 4h in the presence of the proteasome inhibitor MG132. Level of ERK3 and ERK4 proteins were detected by Western blot against GFP or BE. Actin was used as loading control. Figure 3: Specific interaction between MK5 and ERK4. A) GST pull down using recombinant GST, GSTMK2 and GST-MK5 and lysate of E.coli expressing His-ERK4. Co-precipitation of His-ERK4 was detected by Western blotting using an anti-His antibody. B) GST pull down from lysates of HEK293 cells co-expressing GFP, GFP-ERK3 or GFP-ERK4 and GST-MK2 or GST-MK5 developed with anti-GFP antibody. C) GSTERK4 pull down of endogenous MK5 from WT and, as control, MK5-deficient (MK5-/-) mouse embryonic fibroblasts (MEFs). After pull down, MK5 was detected by Western blot. D) Co-immunoprecipitation of endogenous ERK4 and MK5 from MEFs of different genotypes. After IP with MK5 antibodies, ERK4 was detected by Western blot. E) GFP tagged C-terminal deletion mutants of ERK4 (GFP-ERK4∆C1-3, cf. Fig. 1B) were transfected together with GST-MK5. After GST pull down anti-GFP Western blot was performed. Figure 4: ERK4 and ERK3 show cytoplasmic localisation and translocate MK5 but not MK2. Confocal fluorescence microscopy of GFP, GFP-ERK4, and GFP-ERK3 in HEK293 cells demonstrates mainly cytoplasmic localisation of ERK4 and ERK3 (left panel). GFP-MK5 but not GFP-MK2 is translocated to the cytoplasmic compartment when coexpressed with GST-ERK4, His-ERK4, GST-ERK3 or His-ERK3 (right panel). Figure 5: MK5 is activated by ERK4. A) Co-expression of GFP-ERK4, -ERK3 and deletion mutants with GST-MK5 in HEK293 cells. MK5 in vitro kinase assay using recombinant Hsp25 as substrate was carried out after GST pull down. Protein phosphorylation was detected by phosphor-imaging. B) Endogenous ERK4 was immunoprecipitated from lysate of MK%-deficient cells, redissolved and analysed for its ability to activate MK5 in a coupled kinase assay using recombinant MK5 and its in vitro substrate Hsp25. A control immunoprecipitation (C) was performed using antibodies prepared from pre-immune serum. C) Co-expression of His-tagged wild type ERK4 and ERK3 and of catalytically dead ATP-binding mutants of ERK4 (K49A, K50A – “ERK4 KK/AA”) and ERK3 (K49A, K50A – “ERK3 KK/AA”) with GFP-MK5. MK5 in vitro kinase assay using recombinant Hsp25 as substrate was carried out after GFP-immunoprecipitation. D) Fluorescence

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LEGENDS TO THE FIGURES

microscopy of cytoplasmic translocation of CFP-MK5 by both GFP-ERK4 and the catalytically inactive mutant ERK4 KK/AA. Figure 6: Activated MK5 phosphorylates ERK4. Different bands for GFP-ERK4 were detected by anti-GFP Western blot. A) A slow migrating band of GFP-ERK4 appears when co-expressed with GST-MK5 in HEK293 cells. Calf intestinal phosphatase (CIP) treatment for different times (10 and 30 min) leads to gradual disappearance of the slower migrating band. B) Slow migrating band of GFP-ERK4 appears when co-expressed with wild type GST-MK5 but not with kinase-dead GST-MK5-K51E in HEK293 cells. C) The slower migrating GFP-ERK4 band does not exist for the kinase dead mutant of GFP-ERK4-KK/AA when co-expressed with GST-MK5 in HEK cells. Figure 7: The MK5 C-terminal region between amino acids 368 and 473 is necessary for interaction with ERK4. A) Schematic structure of MK5. B) GST-ERK4 pull down of GFP-MK5, its C-terminal deletion mutant GFP-MK5-1-368 and as a negative control of GFP-MK3 from cell lysates of co-transfected HEK293 cells. GFPMK5 was detected in pull down and whole lysate by Western blot against GFP. C) Fluorescence microscopy of cytoplasmic translocation of GFP-MK5 by ERK4 which is not observed for GFP-MK5-1-368.

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Figure 8: ERK3 and ERK4 can form homo- and heteromers when co-expressed in HEK239 cells. A) BEERK4 pull down of GST-ERK4 and GST-ERK3 from cell lysate of co-transfected HEK293 cells. B) BE-ERK3 pull down of GST-ERK4 and GST-ERK3 from cell lysate of co-transfected HEK293 cells.

42

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47

III. SUMMARY AND DISCUSSION

48

SUMMARY AND DISCUSSION In the present work we carried out a characterization of atypical protein kinases ERK3 and ERK4 and detailed study of the ERK3/4 signaling module with MK5. Both kinases comprise a family of atypical kinases that exhibits sequence similarity to ERK1/2 (42% sequence identity), but differ in behavior compared to typical MAPKs. Due to their large C-terminal region following the protein kinase domain, ERK3/4 are almost twice the size of ERK1/2. Another remarkable difference between these two families is the variation in the activation loop motif. The dual phosphorylation site TXY between sub-domain VII and VIII of ERK1/2 is not present in ERK3/4 which display the sequence SEG instead. ERK1/2 are known as stable proteins that are regulated via phosphorylation (Zhu et al. 1994; Boulton et al. 1991; Thomas et al. 1992). In contrast, ERK3 is an unstable protein and is regulated by proteasomal degradation (Coulombe et al. 2003). Typical ERKs can be activated by mitogens, growth factors or interleukins. No extracellular stimuli have been described so far for the atypical kinases ERK3/4 that would lead to their activation (Boulton et al. 1991; Thomas et al. 1992; Julien et al. 2003). Consequently, typical ERKs play one of the leading roles in mitogen-induced proliferation by activation of cell cycle regulating proteins like Cyclin D1, c-myc and c-Jun. Transient co-expression of ERK3/4 and MK5 during embryogenesis with a highest peak of expression at embryonic day 11 may indicate their role in development (Turgeon et al. 2000; Schumacher et al. 2004). There are some similarities between the atypical kinases ERK3/4 and typical MAPKs ERK1/2 for example their localization. Both ERK1/2 and ERK3/4 localize to the cytoplasm when over-expressed in cell. Also both groups are able to form homo- and hetero-dimers (Philipova et al. 2005). However, dimerization of ERK1/2 is an activation-dependent process. Here we show that the ERK3/4 family can also make homo- and hetero-dimers or oligomers in a stimulus independent manner as in case of ERK5 (Yan et al. 2001). Proteins from both families can be exported from the nucleus in exportin1/CRM1-dependent manner (Julien et al. 2003). However there are some contradictory reports from different groups concerning localization of ERK3 in non-stimulated cells (Cheng et al. 1996; Julien et al. 2003; Schumacher et al. 2004). Data from our work suggest that ERK3 and ERK4 are mainly cytoplasmic proteins. Accumulation of ERK3/4 in the nucleus after blocking of nuclear export by Leptomycin B is rather slower in comparison with other MAPKs where most of the protein can be observed in the nucleus just 1h after treatment. However ERK3/4 takes more than 3h. Nevertheless the presence of NLS and NES and the ability of Leptomycin B to 49

accumulate both proteins in the nucleus suggests possible nuclear function of ERK3/4. For example, relocalization of MK5 from the nucleus to the cytoplasm when co-transfected with ERK3/4 may be explained by shuttling of ERK3/4 between nucleus and cytoplasm (Schumacher et al. 2004; Kant et al. In press). Earlier it has been described that MK5 can interact with p38 MAPK family kinase and the p38-binding site is in NLS/NES region (Seternes et al. 2002). Both kinases, ERK3 and ERK4 can bind to the C-terminus part of MK5 which lies after p38-binding site. NLS and NES regions of MK5 are located between the kinase catalytic domain of MK5 and ERK3/4 binding site (Schumacher et al. 2004). Here we show that MK5 binds in-between 326 to 340 amino acid region on ERK3/4 that is present just after the kinase domain of ERK3/4. As a result of this interaction with either ERK3 or ERK4, nuclear MK5 is mainly translocated from nucleus to the cytoplasm. It was also clear that ERK3/4 can phosphorylate MK5 at T182 and activation of MK5 is depending on this phosphorylation. After activation of MK5, both ERK3 and ERK4 are phosphorylated and catalytic activity of MK5 is needed for ERK3/4 phosphorylation. Both atypical ERKs show similar expression patterns during mouse embryogenesis with elevated transcript levels in brain, lung and kidney suggesting cooperatively of ERK3 and ERK4 in MK5-activation during development. Furthermore, when co-expressed in HEK293 cells, both ERK3 and ERK4 can be detected in a protein complex together with MK5. In conclusion, it seems that both proteins can phosphorylate each other after interaction and that the active kinase domain of both proteins is required for this phosphorylation-dependent activation. Since the typical for all MAPK kinases dual phosphorylation motif that is responsible for their activation and function is lacking in ERK3/4 family this raises a question about the possibility of existence of any extracellular stimuli for ERK3/4 activation. Presence of ERK3/4 in mammals but not in lower eukaryotes leads to a possibility that they might have lost their dual phosphorylation motif during evolution. However, it seems that there are at least two differences between ERK3 and ERK4. First, the stability of ERK4 protein is significantly higher than that of ERK3. While ERK3 is highly unstable protein mainly regulated at the level of protein stability in a proteasome-dependent manner (Coulomb et al. 2003), ERK4 is highly stable. Structural differences in N-terminus between ERK3 and ERK4, particular at the region that has been described to be responsible for proteasomal degradation may explain the discrepancies in their stability. In our experiments, the differences in protein stability do not depend on the size of the Nterminal fusion used, challenging the finding that ERK3 is degraded after ubiquitinylation of 50

the free N-terminus (Coulomb et al. 2004) and strengthening the notion that the C-terminal part of ERK3 is also involved in targeting degradation (Mikalsen et al. 2005).

ERK4 Signaling

Substrate

Nucleus MK5

MK5 ERK4 MK5

ERK4 ERK3

ERK4 ERK4 ERK4

ERK3 ERK3

X Substrate

Differentiation

26S

Figure 4: Schematic representation of our model of ERK4 signaling module. Generally ERK3/4 is located in cytoplasmic compartment as homo- or hetero- dimer. Normally ERK3 is unstable protein and undergoes proteasomal degradation faster than ERK4. Nuclear export of ERK4 is facilitated by CRM1. Increased level of cytoplasmic ERK4 leads to increased cytoplasmic anchoring of MK5. Interaction between ERK4 and MK5 facilitates activation of both proteins.

Second, we detected differences in the ability of activation for MK5 by catalytically-dead ATP-pocket mutants of ERK4 and ERK3. When co-transfected in HEK293 cells ATP-pocket mutants of ERK3 can still activate MK5 while ERK4 mutants cannot. The ability of ERK3 mutants to activate MK5 obviously depends on the experimental system applied. In transfected HEK293 cells ATP-binding and phosphorylation site mutants can activate MK5 (Schumacher et al. 2004), while use of recombinant proteins expressed in insect Sf-9 cells in in vitro phosphorylation assays and transfected HeLa cells demonstrates a dependence of MK5 activation on catalytic activity of ERK3 (Seternes et al. 2004). Taking into account the 51

above finding that ERK3 and ERK4 can co-exist in protein complexes of transfected cells, the finding that catalytically dead ERK3 activates MK5 can be explained by its ability to recruit active endogenous ERK4 of HEK293 cells to the MK5-containing complex. Since ERK4 is more stable than ERK3, the steady state level of endogenous ERK4 in HEK293 cells is probably higher than that of ERK3. This would also explain that the kinase-dead ERK4 mutant fails to recruit sufficient ERK3 in HEK293 cells to activate MK5. In addition, because of its molar excess, over-expressed kinase-dead ERK4 can also not quantitatively recruit active endogenous ERK4 in this experimental setting. Hence, the seemingly different behavior of ERK3 and ERK4 mutants in activation of MK5 can finally be explained by complex formation and different protein stability of ERK3 and ERK4. In the end, protein stability remains the only clear difference detected for these atypical kinases so far. Proposed scaffold function of ERK3 in ERK3-MK5 interaction was due to large size and non-functional kinase domain of ERK3 protein. For ERK4 protein function presence of ERK3 might be required. After the dimerization between ERK3/4 the complex might be targeted for degradation by ERK3. On the other hand, stability of ERK3 depends on the presence of MK5, which we could clearly show by using MK5 knockout and rescue by MK5. Interaction between these proteins can act in a similar to MK2-p38 module where MK2 is necessary for p38 stabilization. Lacking MK2 in MK2-/- fibroblasts leads to partial knock out of p38 similar to what had been shown in MK5 knock out regarding ERK3 (Kotlyarov et al. 2002; Schumacher et al. 2004). Role of ERK3 in ERK4-MK5 interaction remains unclear. Another question is to understand the mechanism that leads to co-expression of these proteins during development. It might be possible that there are some specific stimuli for MK5 that activates this module during embryogenesis. Here we have shown that the two atypical ERKs, ERK4 and ERK3, share similar properties with respect to pattern of expression, regulation of sub-cellular localization, interaction with and translocation of MK5, and being a substrate for MK5, as well as their difference in stability at protein level and activation of MK5 by their kinase dead mutants. As these two proteins do not belong to typical MAPKs there are still several questions to be answered. One of these important questions to address is about endogenous ERK3/4. Are they also autophosphorylated and activated like ERK7 and do they mainly stay in the nucleus? Since this pathway might be one of developmental pathways, it is useful to know the ways, in which it can be controlled and some upstream kinase or receptor that can activate these enzymes. Moreover, the transient expression of ERK3/4 and MK5 during embryogenesis intrigues about the mechanisms those can regulate this expression pattern. In 52

the past there was a report suggesting that ERK3 can be controlled by p38 pathway on transcriptional level (Zimmermann et al. 2001), thus it would be interesting to check transcriptional regulation of ERK3/4 during embryogenesis of p38 knock-out mice. What are the mechanisms of ERK3 stabilization in some of the differentiated cells? Is the dimerization a prerequisite for ERK4 degradation and further control mechanisms? Does the phosphorylation at the activation loop have any link to stability of protein and is MK5 in any way involved in this process, since it can bind and phosphorylate both enzymes. These questions can be answered in detail by analysis of ERK3/4 knock-out mice. Some further questions have to be answered are: Why both proteins expression is maximal in brain? Are they playing a significant role in brain development or learning? It is also not clear whether ERK4 expression is increased after differentiation like ERK3 in PC12 cells (Coulombe et al. 2003). Can ERK4 also hamper the cell cycle? A model to investigate the contribution of ERK3/4 together with MK5 to development and cell cycle would be conditional and double ERK3/4 knock-out mice. Alternatively, the role of MK5 in ERK3/4 interactions could be explored by using siRNA to silence ERK3/4 in different cell types from MK5 knock-out animals. In order to identify possible upstream kinases that may also lead to understanding of extracellular stimuli as well as to identification of other substrates for ERK3/4, screening systems like yeast two hybrid or Ras recruitment could be very useful. Another approach to identify interacting proteins from the mammalian cell through proteomics. Screening for gene expression profile at the time point with maximum expression level of ERK3/4 as well as MK5 may help in discovering some of the target genes for this pathway.

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of

p38

mitogen-activated

protein

kinase

bind

in

the

ATP

site.

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ABBREVIATIONS

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ABBREVIATIONS Ab aa Ala A Amp. APS ATP BMK1 bp C ca. cAMP CDD CIP CREB DAG (d)dNTP DMEM DMSO DTT E. coli ECL EDTA e.g. EGF ERKs FCS GDP GTP His IL IP JIP JNK kb kbp kDa Leu LiAc LMB MAPK MAPKAPK MAPKK MAPKKK MEF MEK

Antibody Amino acid Alanine Ampicillin Ammoniumpersulphate Adenosintriphosphate Big MAPK1 Base pairs Concentration circa Cyclic adenosinmonophosphate Common docking domain Calf intestinal phosphatase cAMP-response element-binding protein Diacylglycerol (Di)Desoxynukleotidtriphosphate Dulbecco's Modified Eagle Medium Dimethylsulfoxide Dithiothreitol Escherichia coli Enhanced chemoluminiscence Ethylendiamintetraacetate for example (exempli gratia) Epidermal growth factor Extracellular signal regulated kinases Fetal calf serum Guanosindiphosphate Guanosintriphosphate Histidine Interleukin Immunprecipitation JNK interacting protein c-Jun N-terminal kinase kilobases kilobasepairs kilo-Dalton Leucine Lithiumacetate Leptomycin B Mitogen activated protein kinase MAPK activated protein kinase MAPK kinase MAPKK kinase Mouse embryonic fibroblast MAPK/ERK activated kinase 66

MEKK MKs MKK MNK MSK MyoD NES NLS OD PAGE PBS PCR PKC rpm RSK RTK SAPK Ser SH Sos TNFα T Trp Y V v/Vol w WT

MAPK/ERK activated kinase Kinase MAPK activated protein kinases MAP kinase kinase MAPK-interacting kinase Mitogen- and stress-activated protein kinase Myogenic determination gene Nuclear export signal Nuclear localization signal Optical density Polyacrylamide-gelelektrophoresis Phosphate buffered saline Polymerase chain reaction Protein kinase C rounds per minute Ribosomal S6 kinase Receptor tyrosine kinase Stress-activated protein kinase Serine Src homology Son of sevenless Tumor necrosis fector α Threonine Tryptophane Tyrosine Volt Volume Weight Wild type

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ACKNOWLEDGMENT

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ACKNOWLEDGMENT I would like to express my profound sense of gratitude to my respectful guide Prof. Dr. Matthias Gaestel and Dr. Alexey Kotlyarov who nurtured me and guided me towards my scientific career and allowed me to work on my own ideas. I would also like to thank them for their extraordinary direction, constant encouragement, suggestions and extensive but helpful criticism and valuable discussions. I am thankful to Prof. Dr. Dietmar Manstein and Prof. Dr. Thomas F Schulz for being my cosupervisor. I am thankful to Prof. Dr. med. R. E. Schmidt and the MD/PhD program for the amicable scientific environment at the Hannover Medical School in Hannover, Germany. I am extremely grateful to Dr. Susanne Kruse and Frau Marlies Daniel for their timely help and smiled face. I am extremely grateful to the Stefanie Schumacher and Kathrin Lass for their kind help and donation of skill and time to me. I would like to thank my family for all of their love and encouragement. I would like to express my heartfelt thanks to Anastassiia Vertii for her great deal of support, scientific discussion and care she had for me. I would like to extend my sincere thanks to my lab mates and friends who supported me in many ways.

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CURICULLAM VITAE

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M.Sc. – Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625021, INDIA.

Phone +91-612-2584236(R) E-mail: [email protected] [email protected]

Shashi Kant Personal Information

Date of Birth

:

10th July 1976

Father’s name

:

Dr. Janardan Prasad

Mother’s name

:

Mrs. Nilam Prasad

Permanent Address

:

A – 42, Sachiwalaya Colony, Kankarbagh, Patna – 800020.

Education

• •

•

Symposia & Workshop Attended

2002Pursuing the PhD degree in Molecular and Cellular biology in Institute for Biochemistry, Hannover Medical School, Hannover (Germany). 2000-2002 - Has completed the Master’s degree in Biotechnology at the School of Biotechnology, Madurai Kamaraj University, Madurai (India). I have completed the course, and have obtained an aggregate of 66% in the qualifying examinations. 1995 – 2000 - Tirhut College of Agriculture, Dholi Bachelor of Science from Rajendra Agriculture University, Pusa, Samastipur (India). I completed my Bachelor’s degree in Agricultural Sciences, and have obtained 78.4 % in the qualifying examinations.

•

“Discussion meeting on Biomolecular Conformation and Function” at the Indian Institute of Science, Bangalore, on July 26th-27th, 2001.

•

“National symposium on functional Genomics” at Madurai Kamaraj University, Madurai, on February 15th-17th 2002.

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Workshop in the Festsaal of the "Stephansstift" (Kirchröder Str. 44), Friday, April 4th to Saturday, April 5th 2003.

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European Research Training Network Meeting “Modulation of signaling cascade for the treatment for cancer, Diabetes and Inflammation” at Nice France on June 4th-6th 2004.

•

Workshop in the Festsaal of the "Stephansstift" (Kirchröder Str. 44), Friday, March 19th to Sunday, March 21st.

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Research Experience

•

•

I am persuing my PhD at Hannover Medical School entitled “Mechanisms of activation and substrate targeting in the MAP kinase cascades” under the guidence of Prof. M. Gaestel, Institute for Biochemistry, Hannover Medical School, Hannover. Work in brief: MAPK is regulate many machanism in the cell from cell cycle regulation, differenciation to apoptosis and others. My work envolve the characterization of ERK4, a core protein of relatively not known member in MAPK as well as other proteins down stream of p38 MAPK pathway. I have pursued a one-year project entitled “Cotransformation of multiple T-DNAs: Segregation analysis in the T1 generation”, under Prof. K Veluthambi, Center for Plant Molecular Biology, at Madurai Kamaraj University, Madurai. Work in brief: My work has dealt with the production of marker free plants; using Agrobacterium based co – transformation methods, using tobbaco (Nicotiana tabacam) as the model system for the work.

•

I have undergone two-month summer training during the period May – July 2001, at the Indian Institute of Science, Bangalore, under the guidance of Prof. S. Ramakumar, Bioinformatics center, Indian Institute Of Science, Bangalore. Work in brief: I worked on the bioinformatics based approach to detect novel protein domains in prokaryotes.

•

I worked on the “Breeding and Agronomical Aspects of Fruit and Vegetable Crops” with Dr. P.K. Singh at Banana Research Station, Hajipur, India, as part of my B.Sc. degree dissertation. Work in brief: The work was oriented in the collection of germplasm of the banana verities and in crop improvement.

Publications

1.

S. Kant, S. Schumacher, M. K. Singh, A. Kispert, A. Kotlyarov and M. Gaestel., Characterization of the atypical MAP Kinase ERK4 and its activation of the MAPK-activated protein kinase MK5. J Biol Chem (In press) 2. S. Schumacher, K. Laaß, S. Kant, Y. Shi, A. Visel, A.D. Gruber, A. Kotlyarov and M. Gaestel., Scaffolding by ERK3 regulates MK5 in development. EMBO-J. 2004 Dec 23, 4770-4779. 3. Kant S, Bagaria A, Ramakumar S., Putative homeodomain proteins identified in prokaryotes based on pattern and sequence similarity. Biochem Biophys Res Commun. 2002 Nov 29;299(2):229-32.

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Extracurricula • r Activities • • •

Computer Experience

•

•

Additional Qualifying Examination

I was part of my college football and volleyball teams during my undergraduate studies, and has participated in many inter-college competitions. I am a life member of the Red Cross Society of India. I was an active member of the National Cadet Corps, Tirhut College of Agriculture, Rajendra Agriculture University, PUSA, Samastipur, Bihar, India. Volunteer of National Service Scheme.

I am well versed in computers, and have a good working knowledge of many bioinformatics related packages like GCG version 10, Insight I. I have a thorough working knowledge of other programs like FASTA, BLAST, Interpro. I have an adequate knowledge of Unix and C programming.

Has scored 94.6 percentile in GATE (Graduate aptitude test in Engineering) held during year 2002. Has scored 3rd rank in the entrance exam conducted by JNU for admission to M.Sc in environmental sciences(2002) from the School of Environmental Sciences, JNU, New Delhi.

Scholarships Received

Has received DBT (Department of Biotechnology) studentship for 2 years during my M.Sc. Has received educational stipend during my B.Sc. under rural experience program.

Languages

I can read, write and speak in English and Hindi.

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Declaration

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Declaration Herewith, I confirm that I have written the present PhD thesis myself and independently, and that I have not submitted it at any other university worldwide.

Hannover, (September 2006) Shashi Kant

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