Adaptor Protein in the c-jun N-Terminal Kinase Signaling Pathway

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1 REFERENCES CONTENT ALERTS Role of the Caenorhabditis elegans Shc Adaptor Protein in the c-jun N-Terminal Kinase Signaling Pathway Tomoaki Mizuno, Kota Fujiki, Aya Sasakawa, Naoki Hisamoto and Kunihiro Matsumoto Mol. Cell. Biol. 2008, 28(23):7041. DOI: /MCB Published Ahead of Print 22 September Updated information and services can be found at: These include: This article cites 31 articles, 13 of which can be accessed free at: Receive: RSS Feeds, etocs, free alerts (when new articles cite this article), more» Downloaded from on April 21, 2014 by PENN STATE UNIV Information about commercial reprint orders: To subscribe to to another ASM Journal go to:

2 MOLECULAR AND CELLULAR BIOLOGY, Dec. 2008, p Vol. 28, No /08/$ doi: /mcb Copyright 2008, American Society for Microbiology. All Rights Reserved. Role of the Caenorhabditis elegans Shc Adaptor Protein in the c-jun N-Terminal Kinase Signaling Pathway Tomoaki Mizuno, Kota Fujiki, Aya Sasakawa, Naoki Hisamoto, and Kunihiro Matsumoto* Department of Molecular Biology, Graduate School of Science, Nagoya University, and SORST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya , Japan Received 12 June 2008/Returned for modification 7 July 2008/Accepted 12 September 2008 Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway. Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved in eukaryotic cells and transduce signals in response to a variety of extracellular stimuli. Each pathway is composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) (4, 13). MAPKKK phosphorylates and activates MAPKK, which in turn activates MAPK by dual phosphorylation of threonine and tyrosine residues within a Thr-X-Tyr motif. Three subgroups of MAPKs have been identified: the extracellular signal-regulated kinases (ERK), the c-jun N-terminal kinases (JNK), and the p38 kinases (4, 13). Much has been learned from genetic and biochemical studies of the ERK pathways. In vertebrate cells, Raf MAPKKK triggers the ERK cascade downstream of Ras guanine nucleotide-binding protein, which itself is activated by growth factors that signal through receptor protein tyrosine kinases. Thus, the ERK cascade appears to be a component of various growth-promoting pathways (4, 19, 25). In invertebrates, the corresponding MAPK pathway has been elucidated through the genetic analysis of Caenorhabditis elegans, which has proven to be an excellent organism for the genetic analysis of cell signaling. In C. elegans vulva development, the MAPK pathway mediates the induction of vulval cell fates and includes the factors LIN-45 Raf (MAPKKK), MEK-2/LET-537 (MAPKK), and MPK-1/SUR-1 (MAPK) (6, 27). Recent studies of C. elegans have also revealed a high degree of conservation of JNK and p38 MAPK signaling components between C. elegans and mammals. The C. elegans JNK pathway, composed of JKK-1 MAPKK and JNK-1 MAPK, regulates coordinated * Corresponding author. Mailing address: Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya , Japan. Phone: Fax: g44177a@nucc.cc.nagoya-u.ac.jp. Published ahead of print on 22 September movement via type D GABAergic (GABA, gaminobutyric acid) motor neurons (9) and has a role in synaptic vesicle transport (2). However, it is still unknown which MAPKKK functions upstream of the JKK-1 JNK-1 pathway. C. elegans also possesses another JNK-like MAPK pathway, composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK. KGB-1 has a novel activation site, consisting of Ser-Asp-Tyr rather than Thr-X-Tyr (21, 26). The KGB-1 pathway regulates the stress response to heavy metals (21). The C. elegans PMK-1 p38 MAPK pathway is involved in innate immunity and oxidative-stress response (8, 10). This pathway is composed of NSY-1 MAPKKK, SEK-1 MAPKK, and PMK-1 MAPK. A major question in MAPK cascade signaling is how MAPKKKs can act selectively in their respective MAPK pathways, given that MAPKKKs can phosphorylate and activate multiple MAPKKs. Emerging evidence indicates that this specificity is achieved, in part, by the use of scaffolding or anchoring proteins to coordinate the specific binding of MAPKKKs to individual upstream and downstream MAPKKs. Scaffolding of multicomponent regulatory systems is now recognized as a major mechanism for controlling signal transduction pathways. Components of the JNK pathway can be organized into signaling complexes mediated by a particular scaffold protein(s), for example, members of the JNK-interacting protein (JIP) family (5, 22, 30). In C. elegans, the JIP3 homolog UNC-16 is involved in the JNK-1 pathway, but not in the KGB-1 pathway (2). In this study, we identified SHC-1 as a component of the KGB-1 pathway. SHC-1 is a homolog of the mammalian Shc adaptor and interacts with MEK-1 MAPKK, but not with other MAPKKs, such as JKK-1 or SEK-1. We show that SHC-1 mediates activation of the KGB-1 pathway by connecting MEK-1 with MLK-1 MAPKKK. Our results thus establish a specific genetic link between the Shc adaptor and the JNK pathway. 7041

3 7042 MIZUNO ET AL. MOL. CELL. BIOL. MATERIALS AND METHODS Plasmids. The cdna for shc-1 was isolated by the Y. Kohara EST project (National Institute of Genetics, Mishima, Japan). The mammalian expression vectors for hemagglutinin (HA) epitope-tagged KGB-1 (HA KGB-1) and FLAG epitope-tagged MEK-1 (FLAG MEK-1) were described previously (21). The mammalian expression constructs for FLAG JKK-1, FLAG SEK-1, and T7 SHC-1 were constructed by inserting each coding sequence into a vector expressing epitope-tagged protein under the control of the cytomegalovirus promoter. Each coding sequence was amplified by PCR using primer sets to create restriction sites immediately before the first codon and after the stop codon. Mutated forms of SHC-1 were made by oligonucleotide-directed PCR, and the mutations were verified by DNA sequencing. Gateway cloning technology (Invitrogen) was used to construct the shc-1p::venus, mek-1p::venus, mlk-1p:: venus, dpy-30p::venus, shc-1p::flag::shc-1::venus, dpy-7p::flag::shc-1::venus, dpy- 7p::t7::shc-1, mek-1p::mek-1::venus, dpy-7p::mek-1::venus, and dpy-7p::ha::mlk-1:: venus plasmids for expression in worms. The shc-1p::venus, mek-1p::venus, and mlk-1p::venus plasmids were constructed by fusion of the venus coding sequence to 2.7-kbp, 2.8-kbp, and 2.9-kbp genomic fragments containing the shc-1, mek-1, and mlk-1 promoters, respectively. The shc-1p::flag::shc-1::venus plasmid was constructed by fusing four DNA fragments in the following order: a genomic fragment containing the shc-1 promoter, flag, shc-1, and venus coding sequence. The mek-1p::mek-1::venus plasmid was constructed by connecting three DNA fragments in the following order: a genomic fragment containing the mek-1 promoter, mek-1, and venus coding sequence. The dpy-7p::ha::mlk-1::venus plasmid was constructed by connecting four DNA fragments in the following order: a 400-bp genomic fragment containing the dpy-7 promoter, ha, mlk-1, and venus coding sequence. The dpy-30p::venus plasmid, which expresses Venus from embryogenesis, was constructed by connecting a 1.5-kbp genomic fragment containing the dpy-30 promoter and the venus coding sequence. Antibodies. Anti-KGB-1 and anti-pmk-1 rabbit polyclonal antibodies were described previously (8, 21). Anti-phospho-KGB-1 rabbit polyclonal antibody was raised against a synthetic phosphopolypeptide, CTSMRM(p)SD(p)YV VTRY [(p), phosphorylated], which corresponds to the activation loop of KGB-1, and affinity purified. Anti-MEK-1 rabbit polyclonal antibody was raised against the synthetic polypeptide CPQPAPHHPSRSSNDHNV, which corresponds to the N-terminal portion of MEK-1, and affinity purified. Anti-HA monoclonal antibody 16B12 (Covance), anti-flag monoclonal antibody M2 (Sigma), anti-t7 monoclonal antibody (Novagen), and anti-phospho-p38 MAPK monoclonal antibody 28B10 (Cell Signaling) were used. C. elegans strains. All C. elegans strains were maintained on nematode growth medium (NGM) plates at 20 C and fed with bacteria of the OP50 strain, as described previously (1). The alleles used in this study were N2 Bristol as the wild type and kgb-1(km21), mek-1(ks54), mlk-1(km19), jkk-1(km2), pmk-1(km25), sek-1(km4), mek-2(q425), shc-1(tm1729), and shc-1(ok198). Strains carrying the shc-1p::venus, mek-1p::venus, or mlk-1p::venus transgene were generated by injecting DNA with the prf4 plasmid, which contains rol-6(su1006), as a dominant genetic marker, into the gonads of young adult N2 animals as described previously (20). Strains carrying the shc-1p::flag::shc-1::venus, dpy-7p::flag::shc-1:: venus, dpy-7p::t7::shc-1, mek-1p::mek-1::venus, dpy-7p::mek-1::venus, or dpy- 7p::ha::mlk-1::venus transgene were generated by injecting DNA with the dpy- 30p::venus marker. Stress sensitivity. Assays for heavy metal and tunicamycin toxicities were carried out as follows. Worms were grown and allowed to lay eggs on NGM plates seeded with bacteria of the OP50 strain. Embryos were transferred to NGM plates or NGM plates containing 100 M copper sulfate, 100 M cadmium chloride, or 1 g/ml tunicamycin. To investigate the effects of transgenes on heavy metal toxicity, embryos expressing Venus were transferred. After incubation for 1 day at 20 C, the numbers of hatched embryos were determined by counting unhatched embryos. The worms that developed into adulthood were counted 4 days after the eggs were laid. The percentage of adults was calculated by multiplying the number of adults by 100 and dividing by the number of hatched worms. To assay for arsenite toxicity, well-fed young adults were picked and transferred to NGM plates or NGM plates containing 5 mm sodium arsenite. After incubation for 1 day, the surviving worms were counted. To determine the activation of KGB-1 by heavy metal stress, animals grown on NGM plates were transferred to 1.5-ml test tubes in H 2 O. The worms were then incubated with H 2 O or 1 mm copper sulfate for 1 h at 20 C and subjected to Western blotting using anti-kgb-1 and anti-phospho-kgb-1 antibodies. Peptide synthesis and association assays. Peptides used for binding assays were synthesized and conjugated to beads by Operon Biotechnologies. The peptide-conjugated beads were incubated with cell lysates for 2 h at 4 C and washed three times. The sequence of the MLK-1 peptide used is VQNPQ(p)Y IQCKK. RESULTS SHC-1 associates with MEK-1. The C. elegans interactome project reported that SHC-1 binds to MEK-1 MAPKK, which functions in the KGB-1 pathway (16). In mammals, members of the Shc family of adaptor/docking proteins are important components of signaling pathways induced by various extracellular signals, such as growth factors, cytokines, and various stresses (24). Members of the Shc family of proteins contain an N-terminal phosphotyrosine binding (PTB) domain and a C- terminal Src homology 2 (SH2) domain (23, 24). The C. elegans SHC-1 protein has a domain organization similar to that of mammalian Shc (Fig. 1A). To confirm the interaction between SHC-1 and MEK-1, we conducted immunoprecipitation assays using mammalian HEK293 cells. T7-tagged SHC-1 and FLAGtagged MEK-1 were coexpressed in HEK293 cells. We immunoprecipitated T7 SHC-1 with T7 antibodies and probed for FLAG MEK-1 on a Western blot with anti-flag antibodies. We found that SHC-1 coimmunoprecipitated with MEK-1 (Fig. 1B, line 2), indicating that SHC-1 can physically associate with MEK-1. We then asked whether the catalytic activity of MEK-1 affects this interaction. T7 SHC-1 was coexpressed with a catalytically inactive form of MEK-1 [MEK-1(K99R)] in HEK293 cells. We found that SHC-1 associated with MEK-1 independently of the catalytic activity of MEK-1 (Fig. 1C, line 4). Next, we asked whether SHC-1 associates generally with MAPKKs. We examined the interaction of SHC-1 with JKK-1 and SEK-1, which act upstream of JNK-1, JNK MAPK, and PMK-1 p38 MAPK, respectively (8, 9, 10). T7 SHC-1 was coexpressed with FLAG JKK-1 or FLAG SEK-1 in HEK293 cells and subjected to immunoprecipitation, but no coimmunoprecipitation with JKK-1 or SEK-1 was found (Fig. 1B, lines 4 and 6). Thus, SHC-1 interacts specifically with MEK-1 MAPKK. Since MEK-1 functions as a MAPKK in the KGB-1 pathway, we tested whether SHC-1 also associates with KGB-1 MAPK, but found no such association (Fig. 1D, line 4). SHC-1 contains PTB and SH2 domains in its N- and C-terminal halves, respectively (Fig. 1A). By coimmunoprecipitation assay, we determined that the N-terminal half of SHC-1 (amino acids 1 to 181) containing the PTB domain was sufficient for its interaction with MEK-1 (Fig. 1E, line 3). SHC-1 is specifically involved in the KGB-1 pathway. The above-mentioned binding studies of SHC-1 with different MAPKKs suggested that SHC-1 does not participate in the JNK-1 or PMK-1 pathway in C. elegans. To confirm this possibility, we compared the phenotypes observed in animals harboring the shc-1 deletion mutation (Fig. 1A) with those harboring the jkk-1 or sek-1 mutants. The shc-1 deletion mutants are generally healthy, grow at a normal rate, and produce normal numbers of offspring. The jkk-1(km2) and sek-1(km4) mutant animals exhibit loopy and arsenite-sensitive phenotypes, respectively (8, 9). However, shc-1(tm1729) mutants showed neither phenotype (Fig. 2A and 3 A). Furthermore, the shc-1 mutation had no effect on vulva development (Fig. 2B), which is regulated by another MAPK pathway composed of MEK-2 MAPKK and MPK-1 ERK MAPK (12, 14, 31, 32). These results suggest that SHC-1 is not involved in the JNK-1 JNK, PMK-1 p38, or MPK-1 ERK MAPK pathway.

4 VOL. 28, 2008 ROLE OF C. ELEGANS SHC IN JNK SIGNALING 7043 FIG. 1. SHC-1 interacts with MEK-1. (A) Schematic representation of the structures of the SHC-1 protein and the shc-1 gene. (Top) Hatched and dark boxes represent the PTB and SH2 domains, respectively. The percentages of amino acid similarity in PTB and SH2 domains are also shown in boxes. Comparisons of PTB and SH2 domains between SHC-1 and mouse ShcA are shown. Identical and similar residues are highlighted with black and gray shading, respectively. Essential Arg residues required for binding to phosphotyrosine in PTB and SH2 domains are indicated by asterisks. The bold lines underneath show the extent of the tm1729 and ok198 deletions. (Bottom) Exons and introns are indicated by boxes and lines, respectively. Hatched and dark boxes show PTB and SH2 domains, respectively. The bold lines underneath indicate the extent of the deleted region in each deletion mutant. a.a., amino acids. (B to E) Interaction of SHC-1 with C. elegans MAPKKs and KGB-1. HEK293 cells were transfected with expression vectors encoding T7 SHC-1, FLAG MEK-1 (M), FLAG JKK-1 (J), and FLAG SEK-1 (S) (B); FLAG MEK-1 (wild type [WT]) and FLAG MEK-1(K99R) (KR) (C); HA KGB-1 (D); and T7 SHC-1 (WT), T7 SHC-1 (N), and T7 SHC-1 (C) (E), as indicated. (E) Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-t7 antibodies (IP) were analyzed by Western blotting (WB). A schematic representation of the truncated forms of SHC-1 is also shown. The hatched and dark boxes represent the PTB and SH2 domains, respectively.

5 7044 MIZUNO ET AL. MOL. CELL. BIOL. FIG. 2. Phenotypes of shc-1 mutants. (A) Movement determined in population assay. Well-fed young adults of each animal were spotted in the centers of normal plates and then killed by chloroform 4 min after being spotted. The percentages of worms located outside the 2-cm circle are shown with standard errors. The numbers (n) of animals examined are shown below. WT, wild type. (B) Vulva development. Nomarski images of young adult stage animals are shown. The vulvas are indicated by arrowheads. The bracket indicates the midbody region, in which the vulva is normally induced. The asterisks indicate developing embryos. The scale bars represent 100 m (lef) and 10 m (righ). Since the KGB-1 pathway regulates the response to heavy metal stress (21), we tested whether SHC-1 also regulates a stress response to heavy metals. Animals carrying the shc- 1(tm1729) or shc-1(ok198) mutation (Fig. 1A) were placed on agar plates containing heavy metal ions, and their development FIG. 3. Stress sensitivity in shc-1 mutants. (A) Arsenite sensitivity. Well-fed young adults of each animal were transferred to normal plates containing 5 mm sodium arsenite (As). The percentages of worms surviving after incubation for 1 day are shown with standard errors. WT, wild type. (B) Tunicamycin sensitivity. Each animal was cultured from embryogenesis on normal plates containing 1 g/ml tunicamycin (Tm). The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. FIG. 4. Heavy metal stress sensitivity in shc-1 mutants. Each animal was cultured from embryogenesis on normal plates containing 100 M copper ion (A and C) or 100 M cadmium ion (B). The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. WT, wild type. was monitored for any signs of an altered response to these toxic compounds. We found that shc-1 mutants were hypersensitive to copper (Cu 2 ) and cadmium (Cd 2 ) ions (Fig. 4A and B). The shc-1 mutants grew poorly on plates containing Cu 2 (100 M) or Cd 2 (100 M), whereas N2 wild-type animals grew well, becoming adults within 4 days. In fact, most of the mutant animals failed to reach the adult stage within 4 days. These results indicate that SHC-1 is involved in the regulation of the response to heavy metal stress. In addition, we found that this heavy-metal-sensitive phenotype in shc- 1(tm1729) mutants was suppressed by overexpression of the mek-1 gene (Fig. 4C), suggesting that SHC-1 functions upstream of MEK-1 in the KGB-1 pathway. We compared the expression patterns of the shc-1 and mek-1 genes by fusing their respective promoters to venus to generate shc-1p::venus and mek-1p::venus. The shc-1 and mek-1 genes are expressed ubiquitously in tissues such as the hypodermis, intestine, pharynx, neurons, and body muscles (Fig. 5A and B). Similar expression patterns were observed in transgenic animals harboring the mlk-1p::venus reporter (Fig. 5C). To test whether expression of SHC-1 in the hypodermis of shc-1 mutants confers resistance to heavy metal stress, we expressed the shc-1 cdna in the hypodermis using the dpy-7 promoter. The shc-1(tm1729) deletion mutant carrying dpy-7p::flag::shc-1:: venus expressed SHC-1 in the hypodermis (data not shown) and exhibited resistance to heavy metal stress (Fig. 5D). Similarly, when MEK-1 was expressed in the hypodermis by the dpy-7 promoter, the mek-1 defect was rescued (Fig. 5E). These

6 VOL. 28, 2008 ROLE OF C. ELEGANS SHC IN JNK SIGNALING 7045 Downloaded from FIG. 5. Expression of shc-1 in the hypodermis determines resistance to heavy metal stress. (A to C) Expression patterns of the shc-1p::venus (A), mek-1p::venus (B), and mlk-1p::venus (C) constructs. Hypodermal expression of each construct is shown in below. The arrowheads indicate nuclei of hypodermal cells. (D and E) Heavy metal stress sensitivity. Each animal was cultured from embryogenesis on normal plates containing 100 M copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. WT, wild type. (F) Interaction of SHC-1 with MEK-1 in C. elegans. Extracts were prepared from N2 (WT) and shc-1(tm1729), mek-1(ks54);shc-1(tm1729), and mlk-1(km19);shc-1(tm1729) mutant animals harboring the dpy-7p::t7::shc-1 transgene as an extrachromosomal array (Ex). Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-t7 antibodies (IP) were analyzed by Western blotting (WB). The arrow indicates the position of MEK-1. results suggest that SHC-1 and MEK-1 function in the hypodermis to confer resistance to heavy metal stress. We next examined complex formation between SHC-1 and MEK-1 in animals. We generated a worm strain expressing T7-tagged SHC-1 under the control of the dpy-7 promoter by injecting the dpy-7p::t7::shc-1 transgene. The dpy-7p::t7::shc-1 fusion gene fully rescued the heavy-metal-sensitive phenotype of shc-1 mutants (data not shown), indicating that it is functional in vivo. Lysates from animals were immunoprecipitated with T7 antibodies, and endogenous MEK-1 was detected by immunoblotting. We found that SHC-1 coimmunoprecipitated with MEK-1 (Fig. 5F, line 2), and this interaction was still detected in the absence of MLK-1 (Fig. 5F, line 4). These results suggest that SHC-1 constitutively forms a complex with MEK-1. We investigated whether SHC-1 is involved in the activation of KGB-1 in C. elegans. KGB-1 is activated by MEK-1-mediated phosphorylation at the Ser-198 and Tyr-200 residues in kinase domain VIII of KGB-1 (21). To assay the activation of KGB-1 in animals, we prepared anti-phospho-kgb-1 antibodies that recognize the phosphorylated form of KGB-1. We first confirmed that this antibody could be used to monitor KGB-1 activation in C. elegans. Western blot analysis with the antiphospho-kgb-1 antibody showed that we could detect the phosphorylated form of KGB-1 in wild-type animals (Fig. 6A, line 1). The KGB-1 pathway regulates the response to heavy metal stress (21), and we found that we could upregulate KGB-1 activity by treatment of animals with Cu 2 ion (Fig. 6A, line 2). Animals harboring the kgb-1 deletion mutation exhibited diminished levels of KGB-1 protein and KGB-1 activation (Fig. 6A, lines 3 and 4). In animals harboring mek-1 deletion mutations, KGB-1 activity was markedly reduced compared with wild-type animals (Fig. 6A, lines 5 and 6). To examine the role of SHC-1 in the activation of KGB-1, we tested deletion mutant alleles of the shc-1 gene. We found that phosphorylated KGB-1 levels were significantly decreased in shc-1 deletion mutants, whether treated with Cu 2 or not (Fig. 6A, lines 7 to 10). These results indicate that SHC-1 is required for the on April 21, 2014 by PENN STATE UNIV

7 7046 MIZUNO ET AL. MOL. CELL. BIOL. FIG. 6. Effects of the shc-1 mutation on KGB-1 and PMK-1 activities. (A) Effects of the shc-1 mutation on KGB-1 activity. N2 (wild type [WT]), kgb-1(km21), mek-1(ks54), shc-1(tm1729), and shc- 1(ok198) animals were treated with or without copper ion. Extracts prepared from each animal were immunoblotted with anti-phospho- KGB-1 (P-KGB-1) and anti-kgb-1 antibodies. (B) Effects of the shc-1 mutation on PMK-1 activity. Extracts prepared from each animal were immunoblotted with anti-phospho-p38 MAPK (P-PMK-1) and anti- PMK-1 antibodies. The arrow indicates the position of PMK-1. activation of KGB-1 in C. elegans. To confirm that SHC-1 is specifically involved in the KGB-1 pathway but not in the PMK-1 pathway, we asked whether the shc-1 mutation affected PMK-1 activation in C. elegans. Western blot analysis using an anti-phospho-p38 antibody (10) that specifically recognizes the phosphorylated, activated form of p38 MAPK revealed that the sek-1 deletion mutation, defective in SEK-1 MAPKK functioning upstream of PMK-1, was defective in the activation of PMK-1 (Fig. 6B, line 2). As observed previously (11), the mek-1 mutation partially decreased the activation of PMK-1 (Fig. 6B, line 4). In contrast, the shc-1 deletion mutation was found to have no effect on PMK-1 activation (Fig. 6B, line 5). Taken together, these results suggest that SHC-1 specifically participates in the KGB-1 signaling pathway. We next examined whether the KGB-1 pathway regulates responses to other types of stress. To explore the effects of endoplasmic reticulum (ER) stress, we examined the effects of tunicamycin. Tunicamycin inhibits glycosylation and causes protein misfolding selectively in the ER (3). We found that kgb-1(km21) mutants were hypersensitive to tunicamycin, similar to the shc-1(tm1729) null allele (Fig. 3B). These results suggest that the KGB-1 pathway and SHC-1 regulate the response to ER stress. The PTB domain of SHC-1 is essential for its function. The SHC-1 protein contains PTB and SH2 domains (Fig. 1A). To test whether these domains are essential for its function, we generated mutations in each. To inactivate the PTB domain, Arg-136 was mutated to Lys (R136K), and to inactivate the SH2 domain, Arg-234 was mutated to Lys (R234K) (Fig. 1A and 7A). A mutant in which both residues were mutated (R136K R234K) was also constructed. Expression of wild-type SHC-1 and SHC-1(R234K) in shc-1 mutants rescued the heavy metal sensitivity (Fig. 7B). On the other hand, the SHC- 1(R136K) mutated form only weakly rescued and the double mutations containing both R136K and R234K failed to rescue the sensitivity to heavy metal stress (Fig. 7B). These results suggest that the N-terminal PTB domain is important for FIG. 7. The PTB domain of SHC-1 is essential for resistance to heavy metal stress. (A) Schematic representation of the mutant forms of SHC-1. Hatched and dark boxes represent the PTB and SH2 domains, respectively. (B and C) Heavy metal stress sensitivity in shc-1 mutants. Each animal was cultured from embryogenesis on normal plates containing 100 M copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. WT, wild type. (D) Interaction of SHC-1 mutants with MEK-1. HEK293 cells were transfected with expression vectors encoding T7 SHC-1 (WT), T7 SHC-1(R136K), T7 SHC-1(R234K), T7-SHC-1(R136K R234K), and FLAG MEK-1 as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-t7 antibodies (IP) were analyzed by Western blotting (WB).

8 VOL. 28, 2008 ROLE OF C. ELEGANS SHC IN JNK SIGNALING 7047 FIG. 8. The tyrosine-phosphorylated NPXY motif on MLK-1 provides a docking site for the SHC-1 PTB domain. (A) Schematic representation of MLK-1. Hatched and dark boxes represent the SH3 and kinase domains, respectively. The sequence alignments of the C-terminal portion of MLK-1 and the consensus binding sequence of the PTB domain are shown. The arrow indicates the Tyr residue required for phosphorylation and binding to the PTB domain. (B) Association of SHC-1 with synthetic peptides. Synthetic peptides corresponding to the NPXY sequence of MLK-1 were incubated with lysates prepared from COS-7 cells expressing T7 SHC-1. py and Y represent peptides phosphorylated at Tyr-940 and unphosphorylated, respectively. (C) Association of SHC-1 and MEK-1 with tyrosine-phosphorylated synthetic peptides. py peptides were incubated with lysates prepared from COS-7 cells expressing FLAG MEK-1, T7 SHC-1 (wild type [WT]), and T7 SHC-1(R136K), as indicated. Whole-cell extracts (INPUT) and proteins bound to py peptides (BOUND) were analyzed by Western blotting (WB). (D) Heavy metal stress sensitivity in mlk-1 mutants. Each animal was cultured from embryogenesis on normal plates containing 100 M copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. SHC-1 function while the C-terminal SH2 domain plays a more minor role. Consistent with this, expression of the N- terminal half of SHC-1 (amino acids 1 to 181) containing the PTB domain was able to rescue the shc-1 defect efficiently, while the same construct carrying the R136K mutation failed to rescue the phenotype (Fig. 7C). The result that MEK-1 can associate with the N-terminal half of SHC-1 (Fig. 1E) raised the possibility that the PTB domain of SHC-1 might be essential for its association with MEK-1. However, we found that the SHC-1(R136K) or SHC-1(R136K R234K) mutated form still interacted with MEK-1 (Fig. 7D, lines 3 and 5). Thus, the defect caused by the R136K mutation in the SHC-1 PTB domain is not related to its ability to interact with MEK-1. The NPXY motif of MLK-1 is essential for its function. The structural similarity of SHC-1 with other Shc family proteins suggests that it might associate with proteins that are tyrosine phosphorylated to mediate signal transduction events. Since the SHC-1 PTB domain is functionally important, proteins that interact with SHC-1 in the KGB-1 pathway may be expected to contain consensus binding sites recognized by PTB domains (28). The mammalian Shc PTB domain recognizes phospho- Tyr sites containing the consensus motif NPX(p)Y, with additional selectivity for Ile or Val at the 5 position relative to the phospho-tyr (15, 29). We identified a potential PTB domainbinding NPXY motif, Asn (937)-Pro-Gln-Tyr (940), in the C-terminal region of MLK-1 (Fig. 8A). The NPXY motif in MLK-1 represents a conventional Shc PTB domain binding site, as it contains a Val residue at the 5 position. These results raised the possibility that the SHC-1 protein may possess the ability to bind to the phospho-tyr motif on MLK-1. To test this possibility, synthetic peptides corresponding to the NPXY region of MLK-1 were used to precipitate SHC-1 from lysates of COS-7 cells transiently expressing T7-tagged SHC-1. We found that MLK-1 peptide associated with SHC-1 in a manner dependent upon tyrosine phosphorylation of the peptide (Fig. 8B, lines 3 and 4). We next examined whether the SHC-1 PTB domain is required for binding with the tyrosinephosphorylated (py) MLK-1 peptide. Disruption of the PTB domain by the SHC-1(R136K) mutation abolished the ability of SHC-1 to associate with the py-mlk-1 peptide (Fig. 8C, line 7). These results suggest that the tyrosine-phosphorylated NPXY motif on MLK-1 provides a docking site for the SHC-1 PTB domain. We examined whether SHC-1 allows MLK-1 and MEK-1 to associate. In the absence of SHC-1, MEK-1 failed to associate with the py-mlk-1 peptide (Fig. 8C, line 5). However, addition of SHC-1 induced the interaction between MEK-1 and the py-mlk-1 peptide (Fig. 8C, line 6). Furthermore, the R136K mutation in SHC-1 abolished the binding of MEK-1 to the py-mlk-1 peptide (Fig. 8C, line 7). These results are consistent with the possibility that SHC-1 acts as a scaffold that brings MLK-1 and MEK-1 together. Finally, we determined whether the NPXY motif of MLK-1 is functionally important in animals. We generated a point mutation (Y940F) that changed the Tyr-940 residue of MLK-1 to phenylalanine. HA- and Venus-tagged wild-type MLK-1 and MLK-1(Y940F) were expressed under the control of the dpy-7 promoter in mlk-1(km19) mutants. Compared to wildtype MLK-1, expression of MLK-1(Y940F) in mlk-1 mutants

9 7048 MIZUNO ET AL. MOL. CELL. BIOL. was less efficient in rescuing the sensitivity to heavy metal (Fig. 8D). This demonstrates that the NPXY motif of MLK-1 is important for its function to confer resistance to heavy metal stress. DISCUSSION MAPK cascades are pivotal signaling modules controlling diverse signal transduction pathways in eukaryotes. A major question in MAPK cascade signaling is how similar components can control different biological responses. It is believed that the specificity for distinct signaling pathways is largely determined by scaffolding proteins and specific MAPKs (5, 22). In this study, we present functional evidence showing that a C. elegans Shc adaptor protein, SHC-1, is an essential component of the KGB-1 JNK-mediated stress response pathway. SHC-1 acts as an adaptor to link MEK-1 MAPKK to MLK-1 MAPKKK in a manner dependent on tyrosine phosphorylation of MLK-1. This is a novel role of the Shc family proteins in transducing intracellular signals. SHC-1 is specifically involved in the KGB-1 pathway. An shc-1 loss-of-function mutation is defective in the activation of KGB-1, resulting in hypersensitivity to heavy metal stress. Coimmunoprecipitation analyses in mammalian cells demonstrated that SHC-1 can bind to MEK-1 MAPKK, an upstream regulator KGB-1 MAPK. We have previously identified other MAPK pathways composed of the JKK-1 MAPKK JNK-1 MAPK and the SEK-1 MAPKK PMK-1 MAPK. SHC-1 is unable to associate with JKK-1 or SEK-1, indicating that SHC-1 specifically interacts with MEK-1. Consistent with this specific association, the in vivo phenotype of the shc-1 mutation was different from those observed with JKK-1 or SEK-1 pathway mutations. For example, the jkk-1 and sek-1 mutants exhibited loopy and arsenite-sensitive phenotypes, respectively (8, 9), while the shc-1 mutants exhibited neither phenotype. Thus, SHC-1 is specifically involved in the KGB-1 pathway, but not in the JNK-1 or PMK-1 pathway. We have previously shown that MEK-1 also acts upstream of PMK-1 (11). Indeed, the mek-1 mutation caused a partial decrease in PMK-1 activity. On the other hand, the shc-1 mutation had no effect on PMK-1 activation. In addition, PMK-1 activity was not affected by disruption of the mlk-1 gene, which encodes a MAPKKK functioning upstream of MEK-1 in the KGB-1 pathway (data not shown). These results suggest that MEK-1 functions in the PMK-1 pathway in a manner independent of SHC-1 or MLK-1. This raises the possibility that the interaction of SHC-1 with MEK-1 may confer specificity on the action of MEK-1 on the KGB-1 signaling pathway by connecting MEK-1 with MLK-1. Function of SHC-1 in the KGB-1 pathway. Shc adaptor proteins are conserved from nematodes to mammals (18). In mammalian cells, Shc proteins function as molecular scaffolds in various signaling pathways, including those mediated by receptor tyrosine kinases, cytokine receptors, and oncogenic tyrosine kinases. For example, ShcA plays an important role in the transduction of signals from the epidermal growth factor receptor (EGFR) to the Ras-ERK MAPK pathway (24). In this signaling pathway, ShcA acts as an adaptor to link EGFR to another adaptor, Grb2. Upon activation and phosphorylation of EGFR, ShcA is recruited to EGFR via its PTB or SH2 FIG. 9. Proposed model for SHC-1 function in the KGB-1 JNK signaling pathway. domain and consequently becomes phosphorylated at the Tyr residues 239, 240, and 317, all of which are located within the region between the PTB and SH2 domains. Both Tyr-239 and Tyr-317 form an optimal binding site for the SH2 domain of Grb2. Grb2, in turn, constitutively associates with Sos, a Ras guanine nucleotide exchange factor, through its SH3 domains. Recruitment of the Grb2-Sos complex by ShcA causes membrane relocalization of Sos, resulting in the activation of Ras and its downstream effectors, including the ERK MAPK cascade. Indeed, Drosophila Shc, named DSHC, has been reported to function in signaling from EGFR to the ERK MAPK cascade (17). In C. elegans, signaling from EGFR to Ras and the ERK MAPK pathway has been analyzed most extensively during the formation of the hermaphrodite vulva (7, 12, 14, 31, 32). Components that are known to function in this signal transduction pathway include LET-23 (EGFR), SEM-5 (Grb2), LET-60 (Ras), LIN-45 (MAPKKK), MEK-2 (MAPKK), and MPK-1 (ERK MAPK). Mutations defective in this pathway result in a vulvaless phenotype (6, 27). However, we observed no obvious defect in vulva development in shc-1 mutants. Consistent with this, SHC-1 has no optimal binding site for Grb2, which is conserved from Drosophila DSHC to mammalian Shc proteins (18). Thus, SHC-1 is not essential for signaling from EGFR to Ras and ERK MAPK. The heavy-metal-sensitive phenotype observed in shc-1 mutants was suppressed by overexpression of the mek-1 gene, suggesting that SHC-1 functions upstream of MEK-1 in the KGB-1 pathway. How does SHC-1 regulate MEK-1? Peptide association analyses demonstrated that SHC-1 can bind to MLK-1 MAPKKK, an upstream regulator of MEK-1, and that it promotes an association of MEK-1 with MLK-1. In particular, the interaction between SHC-1 and MLK-1 depends on phosphorylation of a specific tyrosine residue located in the NPXY motif, the PTB domain consensus-binding site, of MLK-1. Consistent with this, introduction of a mutation into the PTB domain of SHC-1 or the NPXY motif of MLK-1 abrogated the ability to confer resistance to heavy metal stress. These data suggest that tyrosine phosphorylation of MLK-1 in the NPXY motif creates a binding site for SHC-1 via the PTB domain and results in the recruitment of the SHC-1 MEK-1 complex to MLK-1 (Fig. 9). Our results further suggest that C. elegans contains a tyrosine kinase(s) that is involved in the stress-signaling pathway by mediating tyrosine phosphorylation of MLK-1. In this model (Fig. 9), SHC-1 regulates MLK-1- mediated phosphorylation and activation of MEK-1 by bringing MEK-1 into close proximity with MLK-1. Thus, SHC-1 acts as a scaffold between MLK-1 and MEK-1 in the KGB-1 JNK signaling pathway. Scaffold proteins play a crucial role in the MAPK pathway by assembling the components of the MAPK

10 VOL. 28, 2008 ROLE OF C. ELEGANS SHC IN JNK SIGNALING 7049 cascade into a multienzyme complex. This recruitment can modulate the signaling strength of the cognate MAPK module by regulating the amplitude and duration of the transduced signal (5, 22). Our findings therefore demonstrate that SHC-1 is a novel member of the family of scaffold proteins functioning in JNK signaling. Several scaffold proteins, including JNKinteracting protein family proteins, have been reported to interact with components of the JNK MAPK pathway to create functional signaling modules (5, 22, 30). However, there is no evidence that association of these scaffold proteins with their partners requires tyrosine phosphorylation. Thus, SHC-1 appears to be unique in this respect, requiring tyrosine phosphorylation to assemble components of the JNK signaling pathway into a multienzyme complex. In this report, we describe an unexpected association of C. elegans SHC-1 with MEK-1 MAPKK and MLK-1 MAPKKK. 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