THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35511–35519, November 17, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Characterization of the Atypical MAPK ERK4 and Its Activation of the MAPK-activated Protein Kinase MK5*□ S

Received for publication, July 14, 2006, and in revised form, September 7, 2006 Published, JBC Papers in Press, September 13, 2006, DOI 10.1074/jbc.M606693200

Shashi Kant‡, Stefanie Schumacher‡, Manvendra Kumar Singh§, Andreas Kispert§, Alexey Kotlyarov‡, and Matthias Gaestel‡1 From the ‡Institute of Biochemistry and §Institute for Molecular Biology, Medical School Hannover, Hannover 30625, Germany

Mitogen-activated protein kinases (MAPKs)2 represent a family of evolutionary conserved enzymes with a central role in the well characterized MAPK signaling cascades. A wide variety of extracellular stimuli serve as activators of MAPK pathways leading to appropriate responses of cells, such as proliferation, differentiation, growth, and migration. MAPK pathways generally have a three-kinase module architecture by which the signal is transmitted from an upstream kinase to a downstream kinase by sequential phosphorylation. MAPKs comprise four well defined groups (ERK1/2 (1, 2), c-Jun N-terminal kinases, p38s, and ERK5 (BMK) (3)), but additional members including ERK3 (1, 4), ERK4 (p63 MAPK, ERK3-related, ERK3␤, MAPK4, Prkm4) (5), and ERK8 (6) have been identified.

* This work was supported by European Community Grant RTN-HPRN-CT2002-00255 and by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. 1 To whom correspondence should be addressed: Medical School Hannover, Institute of Biochemistry, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel.: 49-511-532-2825; Fax: 49-511-532-2827; E-mail: gaestel. [email protected]. 2 The abbreviations used are: MAPK, mitogen-activated protein kinase; BE, BioEase; ERK, extracellular-regulated kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; MEF, mouse embryonic fibroblast; MK, MAPK-activated protein kinase; CIP, calf intestinal alkaline phosphatase; IP, immunoprecipitation; PBS, phosphatebuffered saline.

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ERK4 (p63 MAPK) was described in humans (5), soon after ERK1, ERK2, and ERK3 were identified (1). Among MAPKs, ERK4 is most closely related to ERK3 displaying 62% overall amino acid sequence identity and 73% within the predicted kinase domain. Both kinases do not contain the highly conserved activation loop (“a-loop”) motif TXY between kinase subdomains VII and VIII that is found in all other MAPKs but possess a SEG sequence instead (Fig. 1A). Even the APE motif of subdomain VIII, which is extremely conserved in other MAPKs, is replaced by an SPR motif in ERK3 and ERK4 (Fig. 1A). ERK4 and ERK3 carry long C-terminal extensions (Fig. 1B). Human Erk4 was mapped on chromosome 18q12–21 (7), and a cDNA for the rat homolog rMNK2 was isolated (8). Stimuli, activators, or relevant substrates of ERK4 have remained elusive, and enzymatic activities of the atypical ERKs have not been well defined so far. Initially the MAPK-activated protein kinase MK5 (9, 10), also known as p38-regulated and -activated kinase (PRAK), was described as a member of the MK family and a downstream target of p38 (for recent reviews see Refs. 11 and 12). Previous data suggested that MK5 is not a physiological substrate for p38 in vivo (13), because the stimuli that activate the p38 pathway fail to activate MK5, and binding of endogenous p38 to MK5 is weaker than interaction of p38 with other established substrates, such as MK2 or MK3. Interestingly, it has been recently demonstrated that MK5 strongly interacts with and is activated by ERK3 (14, 15). In this study, we characterize expression, stability, and protein interaction of the ERK3-related kinase ERK4 and analyze its influence on subcellular localization and activity of MK5.

EXPERIMENTAL PROCEDURES Cloning and Site-directed Mutagenesis—For cloning into pENTR/D-TOPO (Invitrogen), the open reading frame of mouse Erk4 cDNA was amplified from the cDNA clone BC062911 (Open Biosystems) by PCR using the primer pair 5⬘-CAC CAT GGC TGA GAA AGG TGA CTG-3⬘ (forward) and 5⬘-TCA CCA CCT TTC TTT GGA GA-3⬘ (reverse). For cloning into pEGFP-C1, an ERK4 cDNA fragment was digested with EcoRI and BamHI after amplification by PCR using the primer pair 5⬘-CTG AGA ATT CAA TGG CTG AGA AAG GTG ACT GC-3⬘ (forward) and 5⬘-CCC CTG GAT CCC TCA CCA CCT TTC TTT GGA G-3⬘ (reverse). The recombination reaction between the entry clone and the pDEST15, pDEST17, pDEST26, pDEST27, and pcDNA6/BioEase-DEST vectors for GST-, His- and BE-tagged ERK4 expression in bacteria and in mammalian system, respectively, were achieved with the LR Supplemental Material can be found at: http://www.jbc.org/cgi/content/full/M606693200/DC1

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The extracellular-regulated kinase (ERK) 4 (MAPK4) and ERK3 (MAPK6) are structurally related atypical MAPKs displaying major differences only in the C-terminal extension. ERK3 is known as an unstable mostly cytoplasmic protein that binds, translocates, and activates the MAPK-activated protein kinase (MK) 5. Here we have investigated the stability and expression of ERK4 and have analyzed its ability to bind, translocate, and activate MK5. We show that, in contrast to ERK3, ERK4 is a stable protein that binds to endogenous MK5. Interaction of ERK4 with MK5 leads to translocation of MK5 to the cytoplasm and to its activation by phosphorylation. In transfected HEK293 cells, where overexpressed catalytically dead ERK3 is able to activate MK5, catalytic activity of ERK4 is necessary for activation of MK5, indicating that ERK4 directly phosphorylates MK5. Interestingly, ERK4 dimerizes and/or oligomerizes with ERK3, suggesting that overexpressed inactive ERK3 recruits active endogenous ERK4 to MK5 for its activation. Hence, ERK3 and ERK4 cooperate in activation of MK5.

ERK4 Interacts with and Activates MK5

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resuspended in 4⫻ 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 ⫻ 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 1 ml of 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 FIGURE 1. Sequence alignment of mouse MAPKs and schematic representation of the primary structure with 25 ␮l of glutathione-Sephaof ERK4 and ERK3 and their deletion mutants. A, alignment of the sequences of the catalytic domains of rose 4B beads (Amersham Biomouse ERK4 (amino acids 153–218), ERK3 (amino acids 155–220), ERK1 (amino acids 171–235), ERK2 (amino sciences) overnight tumbling at acids 151–215), c-Jun N-terminal kinase 1 (amino acids 155–215), and p38 MAPK␣ (amino acids 154 –212) in the region between subdomains VII and VIII containing the activation loop (a-loop). The identities are shaded. The 4 °C. Protein interactions were presence of an SEG motif in ERK4 instead of TXY that is typical for other MAPKs and SPR in place of APE is marked analyzed by Western blot using 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 GFP (anti-GFP B2, Santa Cruz), shown. BE (Streptavidin-horseradish peroxidase; Invitrogen), His (PentaClonase kit (Invitrogen). C-terminal deletion mutants of ERK4 His, Qiagen), and GST antibodies (B14, Santa Cruz). BioEase Pull-down—For BE pull-down HEK293 cells were (Fig. 1B), ERK4⌬C1, -⌬C2, and -⌬C3, were generated from both pENTR/D-ERK4 and pEGFP-C1-ERK4 by using the for- co-transfected either with pcDNA6/BioEase-ERK4 or -ERK3 ward primer as mentioned above for full-length ERK4 and dif- and pDEST27-ERK3 or -ERK4 vectors, after 16 h the cells were ferent reverse primers: ERK4⌬C1, 5⬘-CTT GCG GAT CCA lysed, and supernatant was applied to pull-down with 25 ␮l of TCC TCA CGA GAC ACA GGG T-3⬘; ERK4⌬C2, 5⬘-TCT Streptavidin-agarose beads (Invitrogen). Western blot was GGG ATC CTC ACA TCA GCA CGA TGT CGT CGA-3⬘; developed with anti-GST antibody. ERK4⌬C3, 5⬘-TGC GGG ATC CTC ATT GTG AGG TGG GST-ERK4 Pull-down of Endogenous MK5—5 ⫻ 106 WT and GCT CAT CCT- 3⬘. MK5⫺/⫺ mouse embryonic fibroblast (MEF) cells were transSite-directed mutagenesis was performed in pEGFP-C1 fected with either GST or GST-ERK4 expressing vector. After ERK4-WT using the QuikChange XL site-directed mutagene- 16 h cells were lysed, and GST pull-down was performed by sis kit (Stratagene). For construction of His-tagged ERK4- using 25 ␮l of glutathione-Sepharose beads. Western blot was K49A, K50A expression vector pEGFP-C1-ERK4-K49A, K50A developed with anti-MK5 antibody (a kind gift from Dr. Sir was cloned into pENTR/D-ERK4 vector as a BstXI fragment. Philip Cohen). Recombination reaction was performed between entry and Immunodetection of Endogenous MK5/ERK4 Complexes— pDEST26 using a LR Clonase kit. ERK3 constructs were 5 ⫻ 106 WT, MK5⫺/⫺, or ERK3⫺/⫺ MEF cells were grown in described elsewhere (14). Most MK5 constructs were a kind gift culture and lysed with kinase lysis buffer for 30 min on ice. IP from Dr. Ole Morten Seternes and described elsewhere (16). of endogenous MK5 was performed by incubation of the 1.5 In Vitro Pull-down—Hexahistidine-tagged ERK4 was mg of protein lysate (150 ␮l) with 4 ␮l of anti-MK5 antibody expressed in bacteria, or the protein isolated from different tis- overnight followed by incubation with 15 ␮l of protein sues of mouse. One mg of bacterial or tissue lysate protein was G-Sepharose (Amersham Biosciences) for 1 h at 4 °C. After incubated with either 0.1 nmol of recombinant GST, GST- five washings of the Sepharose beads with IP buffer, the MK2, or GST-MK5 bound to glutathione-Sepharose 4B (Amer- beads were resuspended and boiled in SDS loading buffer. sham Biosciences). After five washes with IP buffer (1⫻ PBS, 50 Western blot was developed using anti-ERK4 antibody (a mM NaF, 1% Triton X-100, 1 mM Na3VO4), the beads were kind gift from Dr. Ole Morten Seternes).

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FIGURE 2. Comparison of protein stability of ERK4 and ERK3. HEK293 cells were transfected with GFP-tagged (top panel ) and BE-tagged (bottom panel ) ERK3 and ERK4. After 16 h of transfection, the cells were treated with cycloheximide (CHX ) to block protein translation for 2 and 4 h and for 4 h 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 a loading control.

of lysate were loaded onto a SDS gel. Protein amounts were analyzed by Western blotting against GFP or BE.

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 analyze whether ERK4 shows a similar tendency to rapid proteosomal degradation, mouse cDNAs of GFP-ERK3 and GFP-ERK4 were expressed in HEK 293 cells, and proteins were compared for stability. After 24 h, protein biosynthesis was inhibited by blocking translation using cycloheximide treatment for 2 and 4 h. 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 GFP-tagged mouse ERK3 is an unstable protein with a half-life of about 1 h that undergoes proteasome-dependent degradation (Fig. 2). In contrast, there was no significant difference in the protein level of GFP-ERK4 after 4 h of 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 proteins was independent of the respective tag (Fig. 2). JOURNAL OF BIOLOGICAL CHEMISTRY

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Western Blot—The protein concentration was measured using the Bradford assay (Bio-Rad). To equalize amounts of protein 4⫻ Laemmli’s SDS sample buffer (40% glycerol, 4% SDS, 4% ␤-mercaptoethanol, 0.4 M Tris-HCl, pH 6.7, and 2 mg/ml bromphenol blue) was added. The samples were boiled and centrifuged. Soluble protein extract was subjected to 10% polyacrylamide-SDS gels and transferred to Hybond ECL membranes (Amersham Biosciences). The blots were incubated for 1 h in PBS with 0.1% Tween 20 containing 5% powdered skim milk. After three washes with PBS with 0.1% Tween 20, the 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-conjugated secondary antibodies at room temperature. Antigen-antibody complexes were detected with an ECL detection kit (Santa Cruz Biotechnology), and the digital chemiluminescence 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 ␮l of 50% glutathione or protein G-Sepharose suspension (Amersham Biosciences), 2.5 ␮l of buffer (500 mM sodium ␤-glycerophosphate, 1 mM EDTA, pH 7.4), 10 ␮g of substrate recombinant Hsp25 in a final volume of 20 ␮l. Then 5 ␮l of hot ATP mixture (20 mM MgCl2, 0.5 mM ATP, 0.1 ␮l of [␥-33P]ATP) was added, and the reaction mix was incubated for 10 min at 30 °C. Radioactivity incorporated into Hsp25 was quantified by phosphorimaging 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 ⫻ 106 HEK293 cells were transiently transfected by Lipofectamine in accordance to the manufacturer’s protocol (Invitrogen). An equimolar amount was used for each vector. For analysis of subcellular localization, GFP and cyan fluorescent protein expression vectors were transfected, the cells were replated in Chambered cover glass (Labtek, Nunc) and analyzed using a Leica DM IRBE microscope with the Leica TCS confocal systems program or Visitron Systems and SPOT Advanced Programme. Staining of nuclei was performed by adding TO-PRO3 (Molecular Probes) to mounting medium (1:1000). Calf Intestinal Alkaline Phosphatase Treatment—pEGFPC1-ERK4 plasmid was transfected in HEK293 cells. After 16 h of transfection cells were lysed in kinase lysis buffer (20 mM Tris acetate, pH 7.0, 0.1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM ␤-glycerophosphate, 50 mM NaF, 5 mM pyrophosphate, 1% Triton X-100, 1 mM benzamidine, 0.1% ␤-mercaptoethanol, 0.27 M sucrose, and 0.2 mM phenylmethylsulfonyl fluoride). Calf intestinal alkaline phosphatase (CIP) (NEB Biolabs) was used for dephosphorylation. 100 ␮g of lysate protein in 1⫻ 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 h the cells were treated with cycloheximide (100 ␮g/ml; Calbiochem) for indicated the time points with or without MG132 (20 ␮M; Biomol). The cells were lysed in kinase lysis buffer and centrifuged, and equal amounts

ERK4 Interacts with and Activates MK5

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. Because Western blot and in situ hybridization analysis detected co-expression of ERK4 and ERK3 with MK5 in mouse (see supplemental Fig. S1), 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

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a more specific interaction of ERK4 exists with MK5 in vivo. This notion is supported by the co-localization 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 analyzed interaction of endogenous proteins in mouse embryonic fibroblasts of different genotypes (WT, MK5⫺/⫺, and ERK3⫺/⫺) using immunoprecipitation with MK5 antibodies and subsequent Western blot detection of ERK4 in the precipitate (Fig. 3D). ERK4 was co-immunoprecipitated with MK5 in WT and ERK3deficient cells, indicating an ERK3-independent interaction between both proteins. The specificity of the co-immunoprecipitation 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 VOLUME 281 • NUMBER 46 • NOVEMBER 17, 2006

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FIGURE 3. Specific interaction between MK5 and ERK4. A, GST pull-down using recombinant GST, GST-MK2, 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 coexpressing GFP, GFP-ERK3 or GFP-ERK4, and GST-MK2 or GST-MK5 developed with anti-GFP antibody. C, GST-ERK4 pull-down of endogenous MK5 from WT and, as control, MK5-deficient (MK5⫺/⫺) 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. WT, wild type; IP, immunoprecipitation; IB, immunoblot.

ERK4 Interacts with and Activates MK5

region homologous to the MK5 binding site in ERK3 between amino acids 330 and 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, whereas GFP-ERK4⌬C3, which lacks the region between amino acids 326 and 340, fails to bind (Fig. 3E). Hence, the region in ERK4 homologous to amino acids 330 – 340 in ERK3, which is essential for ERK3 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 bands on the filter (not shown). ERK4 Translocates MK5 from the Nucleus to the Cytoplasm—We next analyzed 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 counterstain was performed with the dye TO-PRO3. Overexpressed MK5 alone showed predominantly nuclear localization as expected by the presence of a nuclear localizaNOVEMBER 17, 2006 • VOLUME 281 • NUMBER 46

tion signal (16). In contrast, GFP-ERK4 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 in a region that is homologous to ERK4 (18). Cytoplasmic location of ERK4 results from CRM1-dependent nuclear export because treatment with leptomycin B (10 ng/ml; Sigma) for 8 h caused equal distribution of GFP-ERK4 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, was 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 His-tagged 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 co-expression of neither GST- or His-ERK4 nor ERK3. Hence, ERK4 specifically translocates MK5 into the cytoplasm. This also supports the notion that ERK4 binding is independent of the ERK4 fusion tag and specific for MK5 in vivo. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 4. ERK4 and ERK3 show cytoplasmic localization and translocate MK5 but not MK2. Confocal fluorescence microscopy of GFP, GFP-ERK4, and GFP-ERK3 in HEK293 cells demonstrates mainly cytoplasmic localization of ERK4 and ERK3 (left panel ). GFP-MK5 but not GFP-MK2 is translocated to the cytoplasmic compartment when co-expressed with GST-ERK4, His-ERK4, GST-ERK3, or His-ERK3 (right panel ).

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ated an ATP-binding deficient mutant of ERK4 by site-directed mutagenesis. We replaced the lysine residues at positions 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 FIGURE 5. MK5 is activated by ERK4. A, co-expression of GFP-ERK4, GFP-ERK3, and deletion mutants with ERK4 kinase activity for the cytoGST-MK5 in HEK293 cells. MK5 in vitro kinase assay using recombinant Hsp25 as substrate was carried out after plasmic translocation of MK5. The GST pull-down. Protein phosphorylation was detected by phosphorimaging. B, endogenous ERK4 was immunoprecipitated (IP) from lysate of MK5-deficient cells, redissolved, and analyzed for its ability to activate MK5 in GFP-tagged ATP-binding mutant a coupled kinase assay using recombinant MK5 and its in vitro substrate Hsp25. A control immunoprecipitation of ERK4-KK/AA was expressed in (lane C ) was performed using antibodies prepared from preimmune serum. C, co-expression of His-tagged wild HEK293 cells. Similar to wild type 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 ERK4, this mutant is localized in the carried out after GFP immunoprecipitation. D, fluorescence microscopy of cytoplasmic translocation of cyan cytoplasm and translocated cyan fluorescent protein-MK5 by both GFP-ERK4 and the catalytically inactive mutant ERK4 KK/AA. fluorescent protein-MK5 from the nucleus to the cytoplasm (Fig. 5D). ERK4, but Not the ATP-binding Site Mutant ERK4-K49A, Hence, translocation of MK5 to the cytoplasm alone is not sufK50A, Activates MK5 in HEK293 Cells—ERK3 activates ficient to activate MK5. MK5 Phosphorylates ERK4—When ERK4 and MK5 were coMK5 in HEK293 cells by binding and translocation independent of ERK3 catalytic activity (14). We were interested transfected in HEK293 cells, additional low mobility bands of in whether ERK4 regulates MK5 kinase activity in a similar GFP-ERK4 were observed in SDS-PAGE of cell lysates (Fig. 3, B manner. Protein kinases were expressed in HEK293 cells, and E). Furthermore, phosphate incorporation was found in and an in vitro kinase assay was performed after pull-down of bands corresponding to ERK4 or ERK4⌬C1 in in vitro kinase GST-MK5 using the known in vitro substrate Hsp25. We assay (Fig. 5, A and C), suggesting phosphorylation of ERK4 by detected Hsp25 phosphorylating kinase activity and phos- MK5. To test this hypothesis, GFP-ERK4 was co-expressed phorylated MK5 but also phosphorylated ERK4 and in con- with GST-MK5 in HEK293 cells and subsequently treated with trols phosphorylated ERK3. This indicates the existence of a CIP. After 10 min of treatment, the lower migrating bands productive complex between MK5 and ERK4 and ERK3, began to disappear (Fig. 6A), indicating that they were due to respectively. Both co-expressed GFP-ERK4 as well as GFP- phosphorylation of ERK4. To determine whether MK5 can ERK4⌬C1 activated MK5 (Fig. 5A) and were subsequently directly phosphorylate ERK4, HEK 293 cells were co-transalso phosphorylated by MK5 (see below). The activation of fected with wild-type GFP-MK5 and the kinase-dead mutant MK5 by ERK4 was comparable with the activation by ERK3 GFP-MK5-K51E as a control. The additional low mobility band and its C-terminal deletion mutants ERK3⌬C1 and of ERK4 was observed in the presence of wild-type MK5 but not ERK3⌬C2. Only ERK3⌬C3, which is no longer able to bind in the control (Fig. 6B). Thus, MK5 catalytic activity is necesand to translocate MK5 into the cytoplasm (14), failed to sary for phosphorylation of ERK4 that probably occurs in a direct manner. In a further experiment, we tested whether catactivate MK5 in this assay (Fig. 5A). To see whether endogenous ERK4 is able to active MK5 in alytic activity of ERK4 is required for MK5-dependent phosvitro, we immunoprecipitated ERK4 from MK5-deficient MEFs phorylation of ERK4. GST-MK5 was co-transfected with GFPand assayed the immunoprecipitate in a coupled kinase assay ERK4 or the kinase-dead mutant GFP-ERK4-KK/AA, followed using recombinant MK5 and Hsp25. The IP of ERK4-specific by Western blot against GFP. GFP-ERK4 but not the mutant antibodies but not of a preimmune serum (control) was able to GFP-ERK4-KK/AA was phosphorylated under these conditions (Fig. 6C). Because ERK4-KK/AA is unable to activate phosphorylate and activate MK5 (Fig. 5B). We wondered whether activation of MK5 by ERK4 MK5 (Fig. 5C), it is likely that phosphorylation of MK5 is a depends on the catalytic activity of ERK4. Hence, we gener- prerequisite for phosphorylation of ERK4 by MK5.

ERK4 Interacts with and Activates MK5 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 nuclear localization signal (amino acids 361–364), and MK5–1-368, which contains the nuclear localization signal but lacks the further C terminus (Fig. 7A). After co-transfection of HEK293 cells

FIGURE 6. Activated MK5 phosphorylates ERK4. Different bands for GFPERK4 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. CIP treatment for different times (10 and 30 min) leads to gradual disappearance of the more slowly migrating band. B, slow migrating band of GFP-ERK4 appears when co-expressed with wild type GST-MK5 but not with kinasedead 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.

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 with elevated transcript levels in brain, lung, and kidney, suggesting cooperativity of ERK3 and ERK4 in MK5 activation during development (see supplemental material). Both proteins are subject to exportin1/CRM1-dependent nuclear export and carry a conserved region C-terminal to the catalytic domain that interacts with a sequence C-terminal to the nuclear localization signal and p38-binding site of MK5. The region between amino acids 326 and 340 in ERK4, which is homologous the MK5binding region of ERK3, is also necessary for MK5 binding. Both ERK4 and ERK3 bind to the region of MK5 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 C-terminal to amino acid 368, probGFP-MK5–1-368 and as a negative control of GFP-MK3 from cell lysates of co-transfected HEK293 cells. GFP- ably between amino acids 423 and MK5 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. NLS, nuclear local- 472 (15). As a result of interaction with ERK4 and ERK3, nuclear MK5 ization signal; IB, immunoblot. NOVEMBER 17, 2006 • VOLUME 281 • NUMBER 46

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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. GFP-tagged MK3, a kinase that 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 heterodimers with ERK3, HEK293 cells were co-transfected with GST-ERK4 and GST-ERK3, respectively, and BE-ERK4 plasmids. Then BE pulldown was performed and analyzed by Western blot against GST. Specific binding of GST-ERK4 and GST-ERK3 to BEERK4 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 BE pull-down experiment with BE-ERK3 gave similar results (Fig. 8B). Therefore, ERK3 and ERK4 are likely to exist in multimeric protein complexes. Because BE-ERK3 pull-down also precipitates the C-terminal deletion mutant ERK4-⌬C3 (not shown), MK5-binding and ERK3-binding regions in ERK4 are different.

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HEK293 cells to the MK5-containing complex. Because 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, overexpressed kinasedead ERK4 can also not quantitatively recruit active endogenous ERK4. Hence, the seemingly different behavior 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, FIGURE 8. ERK3 and ERK4 can form homo- and heterodimers when co-expressed in HEK293 cells. A, BE-ERK4 pull-down of GST-ERK4 and GST-ERK3 from cell lysate of co-transfected HEK293 cells. B, BE-ERK3 protein stability remains the only pull-down of GST-ERK4 and GST-ERK3 from cell lysate of co-transfected HEK293 cells. IB, immunoblot. clear difference detected for these atypical kinases so far. is exported to the cytoplasm. After activation of MK5, both Finally, we should mention that the physiological role of the ERK3 and ERK4 are phosphorylated depending on MK5 cata- ERK4/MK5 signaling module is still enigmatic, because extralytic activity. Furthermore, when co-expressed in HEK293 cells, cellular agonists of MK5 have not been identified among the both ERK3 and ERK4 can be detected in an oligomeric protein diverse stimuli analyzed at the cellular level (13).4 Similar to the complex together with MK5. Binding between ERK4 and ERK3 ERK3/MK5 module (14), the ERK4/MK5 module may play a depends neither on the C-terminal extension of ERK4 nor on more prominent role in embryonic or post-natal development. the MK5-binding site, indicating direct interaction of the cata- Compound mouse mutants of the different components of lytic domains. these modules will ultimately provide more detailed insight Despite the many similarities, ERK3 and ERK4 seem to into the physiological role of these atypical ERKs. differ at least in two points. First, ERK4 protein is significantly more stable than ERK3 that is rapidly degraded in a Acknowledgments—We thank Dr. Ole-Morten Seternes (Tromsoe) for proteasome-dependent manner (17). The differences in pro- MK5 constructs, ERK4 antibodies and sharing unpublished results, tein stability are independent of the size of the N-terminal Dr. Sylvain Meloche (Montreal) for ERK3-deficient mouse fibroblasts, tag, challenging the finding that ERK3 is degraded after ubiq- Dr. Sir Philip Cohen (Dundee) for MK5-antibodies, and Kathrin uitinylation of the free N terminus (20) while strengthening Laass (Hannover) for help with the kinase assays. We also express our the notion that the C-terminal part of ERK3 is also involved thanks to the M.D./Ph.D. program “Molecular Medicine” of the Medical School Hannover. in targeting degradation (21). Oligomerization of ERK3 and ERK4 may affect ERK3 and/or ERK4 stability under certain conditions. However, our preliminary experiments analyz- REFERENCES ing the effect of co-expression of ERK4 on protein stability of 1. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., ERK3 in HEK293 cells3 did not reveal significant differences Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663– 675 so far. 2. Johnson, G. L., and Lapadat, R. (2002) Science 298, 1911–1912 Second, catalytically dead ATP pocket mutants of ERK4 and 3. Zhou, G., Bao, Z. Q., and Dixon, J. E. (1995) J. Biol. Chem. 270, ERK3 differ in their ability to activate MK5. Upon co-transfec12665–12669 tion in HEK293 cells, ATP pocket mutants of ERK3 still acti4. Zhu, A. X., Zhao, Y., Moller, D. E., and Flier, J. S. (1994) Mol. Cell Biol. 14, vated MK5 (14), whereas ERK4 mutants failed to do so. The 8202– 8211 ability of ERK3 mutants to activate MK5 obviously depends on 5. Gonzalez, F. A., Raden, D. L., Rigby, M. R., and Davis, R. J. (1992) FEBS Lett. 304, 170 –178 the experimental system applied, because both in an in vitro 6. Abe, M. K., Saelzler, M. P., Espinosa, R., 3rd, Kahle, K. T., Hershenson, phosphorylation assay with recombinant proteins expressed in M. B., Le Beau, M. M., and Rosner, M. R. (2002) J. Biol. Chem. 277, insect Sf-9 cells and in transfected HeLa cells MK5 activation 16733–16743 depends on catalytic activity of ERK3 (15). Taking into account 7. Li, L., Wysk, M., Gonzalez, F. A., and Davis, R. J. (1994) Oncogene 9, that ERK3 and ERK4 co-exist in protein complexes in trans647– 649 fected cells, MK5 activation by catalytically dead ERK3 may be 8. Garcia, J. I., Zalba, G., Detera-Wadleigh, S. D., and de Miguel, C. (1996) Mamm. Genome 7, 810 – 814 explained by its ability to recruit active endogenous ERK4 of

ERK4 Interacts with and Activates MK5 9. New, L., Jiang, Y., Zhao, M., Liu, K., Zhu, W., Flood, L. J., Kato, Y., Parry, G. C., and Han, J. (1998) EMBO J. 17, 3372–3384 10. Ni, H., Wang, X. S., Diener, K., and Yao, Z. (1998) Biochem. Biophys. Res. Commun. 243, 492– 496 11. Roux, P. P., and Blenis, J. (2004) Microbiol. Mol. Biol. Rev. 68, 320 –344 12. Gaestel, M. (2006) Nat. Rev. Mol. Cell Biol. 7, 120 –130 13. Shi, Y., Kotlyarov, A., Laass, K., Gruber, A. D., Butt, E., Marcus, K., Meyer, H. E., Friedrich, A., Volk, H. D., and Gaestel, M. (2003) Mol. Cell Biol. 23, 7732–7741 14. Schumacher, S., Laass, K., Kant, S., Shi, Y., Visel, A., Gruber, A. D., Kotlyarov, A., and Gaestel, M. (2004) EMBO J. 23, 4770 – 4779 15. Seternes, O. M., Mikalsen, T., Johansen, B., Michaelsen, E., Armstrong, C. G., Morrice, N. A., Turgeon, B., Meloche, S., Moens, U., and Keyse, S. M. (2004) EMBO J. 23, 4780 – 4791 16. Seternes, O. M., Johansen, B., Hegge, B., Johannessen, M., Keyse, S. M., and Moens, U. (2002) Mol. Cell Biol. 22, 6931– 6945

17. Coulombe, P., Rodier, G., Pelletier, S., Pellerin, J., and Meloche, S. (2003) Mol. Cell Biol. 23, 4542– 4558 18. Julien, C., Coulombe, P., and Meloche, S. (2003) J. Biol. Chem. 278, 42615– 42624 19. 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 20. Coulombe, P., Rodier, G., Bonneil, E., Thibault, P., and Meloche, S. (2004) Mol. Cell Biol. 24, 6140 – 6150 21. Mikalsen, T., Johannessen, M., and Moens, U. (2005) Int. J. Biochem. Cell Biol. 37, 2513–2520

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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

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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

C

Relative Hsp25 phosphorylation

WT

180 160 140 120 100 80 60 40 20 0

Control

ERK3

ERK3-K49,50A

ERK3-S189A

ERK3-K49,50R

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

301

A

357

ERK3-∆C3

471

ERK3-∆C2 ERK3-∆C1 1

316

720

481

ERK3

Kinase

C-extension

ERK4 1

312

GF P ER K ER 3-∆ K C ER 3-∆ 3 K3 C2 ER -∆ K C1 ER 3-S1 89 K ER 3-K A K3 49, 50 R

B

461 478

GST-MK5 pull-down

Lysate

C

GFP-MK5

GFP-ERK3-∆C1

+ His-ERK3-∆C1

GFP-ERK3-∆C2

+ His-ERK3-∆C2

GFP-ERK3-∆C3

+ His-ERK3-∆C3

D

+ His-ERK4

Co nt r ER ol K3 ER K ER 3-∆ K3 C2 ER -∆C K3 1 -∆ C3 ER K3 ER K4

GFP-ERK4

His-ERK3-∆C1 -

- His-ERK3 - GFP-MK5

- Hsp25

4774 The EMBO Journal VOL 23 | NO 24 | 2004

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

ERK3–MK5 signalling module S Schumacher et al

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).

References Abe MK, Kuo WL, Hershenson MB, Rosner MR (1999) Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth. Mol Cell Biol 19: 1301–1312 Abe MK, Saelzler MP, Espinosa III R, Kahle KT, Hershenson MB, Le Beau MM, Rosner MR (2002) ERK8, a new member of the mitogen-activated protein kinase family. J Biol Chem 277: 16733–16743 Baas AF, Boudeau J, Sapkota GP, Smit L, Medema R, Morrice NA, Alessi DR, Clevers HC (2003) Activation of the tumour suppressor

4778 The EMBO Journal VOL 23 | NO 24 | 2004

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

& 2004 European Molecular Biology Organization

ERK3–MK5 signalling module S Schumacher et al

Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ, Cohen P (1995) Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO J 14: 5920–5930 Boudeau J, Baas AF, Deak M, Morrice NA, Kieloch A, Schutkowski M, Prescott AR, Clevers HC, Alessi DR (2003) MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J 22: 5102–5114 Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65: 663–675 Cheng M, Zhen E, Robinson MJ, Ebert D, Goldsmith E, Cobb MH (1996) Characterization of a protein kinase that phosphorylates serine 189 of the mitogen-activated protein kinase homolog ERK3. J Biol Chem 271: 12057–12062 Coulombe P, Rodier G, Pelletier S, Pellerin J, Meloche S (2003) Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin–proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular differentiation. Mol Cell Biol 23: 4542–4558 Engel K, Kotlyarov A, Gaestel M (1998) Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J 17: 3363–3371 Engel K, Plath K, Gaestel M (1993) The MAP kinase-activated protein kinase 2 contains a proline-rich SH3-binding domain. FEBS Lett 336: 143–147 Engel K, Schultz H, Martin F, Kotlyarov A, Plath K, Hahn M, Heinemann U, Gaestel M (1995) Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif. J Biol Chem 270: 27213–27221 Gonzalez FA, Raden DL, Rigby MR, Davis RJ (1992) Heterogeneous expression of four MAP kinase isoforms in human tissues. FEBS Lett 304: 170–178 Herzig U, Cadenas C, Sieckmann F, Sierralta W, Thaller C, Visel A, Eichele G (2001) Development of high-throughput tools to unravel the complexity of gene expression patterns in the mammalian brain. Novartis Found Symp 239: 129–146 Johnson GL, Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298: 1911–1912 Julien C, Coulombe P, Meloche S, Rodier G, Pelletier S, Pellerin J, Turgeon B, Lang BF, Robinson MJ, Xu Be BE, Stippec S, Cobb MH, Zimmermann J, Lamerant N, Grossenbacher R, Furst P (2003) Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression. J Biol Chem 278: 42615–42624 Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, Young PR (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739–746 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934 Marchler-Bauer A, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz DI, Jackson JD, Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler GH, Mazumder R, Nikolskaya AN, Panchenko AR, Rao BS, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Vasudevan S, Wang Y, Yamashita RA, Yin JJ, Bryant SH (2003) CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res 31: 383–387

& 2004 European Molecular Biology Organization

Meloche S, Beatty BG, Pellerin J (1996) Primary structure, expression and chromosomal locus of a human homolog of rat ERK3. Oncogene 13: 1575–1579 Neininger A, Thielemann H, Gaestel M (2001) FRET-based detection of different conformations of MK2. EMBO Rep 2: 703–708 New L, Jiang Y, Han J (2003) Regulation of PRAK subcellular location by p38 MAP kinases. Mol Biol Cell 14: 2603–2616 New L, Jiang Y, Zhao M, Liu K, Zhu W, Flood LJ, Kato Y, Parry GC, Han J (1998) PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J 17: 3372–3384 Ni H, Wang XS, Diener K, Yao Z (1998) MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular-regulated kinase (ERK) and p38 kinase. Biochem Biophys Res Commun 243: 492–496 Papa FR, Zhang C, Shokat K, Walter P (2003) Bypassing a kinase activity with an ATP-competitive drug. Science 302: 1533–1537 Posas F, Saito H (1997) Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276: 1702–1705 Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR (1994) A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027–1037 Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68: 320–344 Saharinen P, Takaluoma K, Silvennoinen O (2000) Regulation of the Jak2 tyrosine kinase by its pseudokinase domain. Mol Cell Biol 20: 3387–3395 Schwarz E, Oesterhelt D, Reinke H, Beyreuther K, Dimroth P (1988) The sodium ion translocating oxalacetate decarboxylase of Klebsiella pneumoniae. Sequence of the biotin-containing alphasubunit and relationship to other biotin-containing enzymes. J Biol Chem 263: 9640–9645 Seternes OM, Johansen B, Hegge B, Johannessen M, Keyse SM, Moens U (2002) Both binding and activation of p38 mitogenactivated protein kinase (MAPK) play essential roles in regulation of the nucleocytoplasmic distribution of MAPK-activated protein kinase 5 by cellular stress. Mol Cell Biol 22: 6931–6945 Shi Y, Kotlyarov A, Laa K, Gruber AD, Butt E, Marcus K, Meyer HE, Friedrich A, Volk HD, Gaestel M (2003) Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination. Mol Cell Biol 23: 7732–7741 Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M (1992) Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313: 307–313 Tanoue T, Nishida E (2003) Molecular recognitions in the MAP kinase cascades. Cell Signal 15: 455–462 Turgeon B, Saba-El-Leil MK, Meloche S (2000) Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa. Biochem J 346: 169–175 Underwood KW, Parris KD, Federico E, Mosyak L, Czerwinski RM, Shane T, Taylor M, Svenson K, Liu Y, Hsiao CL, Wolfrom S, Maguire M, Malakian K, Telliez JB, Lin LL, Kriz RW, Seehra J, Somers WS, Stahl ML (2003) Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme. Structure (Camb) 11: 627–636 Zhou G, Bao ZQ, Dixon JE (1995) Components of a new human protein kinase signal transduction pathway. J Biol Chem 270: 12665–12669 Zhu AX, Zhao Y, Moller DE, Flier JS (1994) Cloning and characterization of p97MAPK, a novel human homolog of rat ERK-3. Mol Cell Biol 14: 8202–8211

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BBRC Biochemical and Biophysical Research Communications 299 (2002) 229–232 www.academicpress.com

Putative homeodomain proteins identified in prokaryotes based on pattern and sequence similarity Shashi Kant,a Ashima Bagaria,b and S. Ramakumarb,c,* a

School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India Department of Physics, Indian Institute of Science, Bangalore 560012, India c Bioinformatics Center, Indian Institute of Science, Bangalore 560012, India

b

Received 13 October 2002

Abstract A putative homeodomain has been identified in eubacterial genomes, which include several pathogens. The domain is related in sequence to homeodomain, a component specific to transcription factors and playing a very important role in eukaryotes such as controlling the developmental processes of the organism. The putative homeodomain has been characterized utilizing the eukaryotic homeodomain protein sequence signature present in PROSITE as well as the sequence similarity search using BLAST suite for different eubacterial genomes. These findings provide evidence for the occurrence of DNA-binding motif in prokarya similar to that in eukarya. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Transcription factors; Homeodomain; Sequence pattern; Homeobox; DNA-binding domain; Prokaryote; Eukaryote; Pro-homeodomain

Homeotic genes—the master control genes that regulate developmental process [1] in a precise spatial and temporal fashion [2] in higher organisms, share a common sequence element, known as homeobox. The homeobox is an important member of homeotic genes and a number of these genes appear to have retained both their precisely ordered tandem arrangement in the genome as well as their developmental roles in axial patterning across vast evolutionary time [2]. It encodes a self-folding, stable protein domain of about 60 amino acids, the homeodomain, which is composed of three helical regions representing the sequence specific DNAbinding domain of much larger transcription factor proteins [2,3]. Homeodomain was first identified in Drosophila homeotic loci, where the proteins play a role in determination of body plan [4]. The sequences related to homeodomain are found in several other organisms, but while moving from the original Drosophila homeodomains to mammalian homeodomains, the relationship between conserved regions drops significantly [5]. * Corresponding author. Fax: +91-80-360-2602/91-80-334-1683. E-mail address: [email protected] (S. Ramakumar).

However, some residues in the sequence are almost conserved, leading to similar structure and function of homeodomain from different organisms [6]. It is a DNAbinding domain with three helices, where the first and the second helices are almost antiparallel to each other and the third helix is almost perpendicular to the other two [7]. The second and the third helices form a helixturn-helix motif [8]. Since the past few years a large number of homeobox genes from taxonomic groups ranging from yeast to human have been isolated. A vast amount of sequence data on homeodomains has been accumulated, which provides useful and important information about the evolution of the homeobox gene family and the phylogeny of eukaryotic organisms [9,10]. On the other hand, a large number of gene sequences either from fully or partially sequenced genomes are available in the public domain. These include genomes from several pathogens as well. However not much is known as to whether proteins similar in sequence and structure to eukaryotic homeodomains occur in prokaryotes also. An attempt to answer this question utilizing currently available bioinformatics tools such as BLAST series of software [11], FASTA [12], CLUSTALW [13], SCAN

0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 6 0 7 - 4