Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway

Abstract

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.

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.

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 μm (lef) and 10 μm (righ).

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 μg/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 μM copper ion (A and C) or 100 μM cadmium ion (B). The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. WT, wild type.

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 μM 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.

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.

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 μM 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).

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 μM copper ion. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors.

FIG. 9.

Proposed model for SHC-1 function in the KGB-1 JNK signaling pathway.