Characterization of shc-1. (A) Alignments of C. elegans SHC-1, human p52Shc, mouse p52Shc, and Drosophila DShc. Identical amino acid residues are highlighted, and similar residues are shaded. The conserved PTB and SH2 domains are underlined. (B) Genomic organization of shc-1 and location of deletion in ok198. Coding regions are indicated by boxes, and introns are represented as lines. SHC-1 has the N- to C-terminal PTB-SH2 modularity of Shc-like proteins (predicted by SMART, Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de). (C) Life-span analysis of shc-1(ok198) and rescue by transgenic expression of human p52shc. All life-span data presented in this and subsequent figures are the result of at least three representative experiments. All life-span data are listed as mean life span ± standard error of the mean; (n) number of animals observed; (Ex) extrachromosomal array; P-values refer to experimental strain and wild-type control animals. shc-1(ok198): 8.9 ± 0.1 d (n = 152, P < 0.0001); wild type: 14.4 ± 0.1 d (n = 204); shc-1(ok198);Ex[p52Shc]: 12.0 ± 0.4 d (n = 93, P = 0.1161 vs. Ex[p52Shc]); Ex[p52shc]: 12.7 ± 0.3 d (n = 82, P < 0.0001). (D–H) shc-1∷gfp is broadly expressed in C. elegans. SHC-1∷GFP stains the pharynx, the majority of head neurons (E), the tail neurons (F), the vulva muscles, and the gonads (G). (H) SHC-1∷GFP localizes to both the cytoplasm and nuclei of intestinal cells. A portion of SHC-1 is localized at the cytoplasmic membrane. The image in D was taken with a 20× objective on a Zeiss Axioplan2 microscope using an EGFP filter set (480/20-nm excitation, 510/20-nm emission). To estimate the autofluorescent background signal, we imaged wild-type worms under the same conditions and detected only a faint yellow fluorescence signal in intestinal cells. Bar, 100 μM. (E–H) Higher-magnification pictures were taken on a confocal laser microscope, always showing L4 larvae. Bars: E–G, 50 μM; H, 25 μM.

shc-1 acts in the IIS pathway for the control of life span and stress response. (A) daf-16(RNAi) does not reduce shc-1 mutant life span. Wild type on pL4440: 15.4 ± 0.4 d (n = 99); shc-1(ok198) on pL4440: 10.8 ± 0.3 d (n = 90, P < 0.0001); daf-16(RNAi): 10.1 ± 0.2 d (n = 100, P < 0.0001); shc-1(ok198); daf-16(RNAi): 10.1 ± 0.3 d [n = 82, P = 0.6713 vs. daf-16(RNAi)]. (B) Life-span analysis of daf-16(mgDf50) and shc-1(ok198). The assays were performed in a fer-15(b26) background at 25°C to prevent progeny. shc-1(ok198); fer-15(b26): 10.5 ± 0.2 d (n = 160, P < 0.0001); fer-15(b26): 14.0 ± 0.2 d (n = 160); daf-16(mgDf50); fer-15(b26): 10.1 ± 0.2 d (n = 107, P < 0.0001); daf-16(mgDf50) shc-1(ok198); fer-15(b26): 10.2 ± 0.3 d (n = 104, P < 0.0001). (C) Overexpression of daf-16 partially suppresses the shc-1 mutant life span. shc-1(ok198): 8.8 ± 0.3 d (n = 70); wild type: 14.4 ± 0.1 d (n = 204); Is[daf-16∷gfp]: 15.8 ± 0.5 d (n = 139, P = 0.0437); shc-1(ok198); Is[daf-16∷gfp]: 11.1 ± 0.4 d (n = 90, P < 0.0001). (D) age-1(hx546) suppresses the shc-1(ok198) aging phenotype. Wild type: 14.2 ± 0.4 d (n = 204); shc-1 (ok198): 8.4 ± 0.3 d (n = 152, P < 0.0001); shc-1(ok198); age-1(hx546): 17.5 ± 0.5 d (n = 178, P < 0.0001); age-1(hx546): 17.6 ± 0.5 d (n = 245, P < 0.0001). (E) daf-2(e1368) extends the short life span of shc-1(ok198). Wild type: 14.3 ± 0.35 d (n = 104); shc-1 (ok198): 10.6 ± 0.3 d (n = 103, P < 0.0001); daf-2(e1368): 25.3 ± 0.6 d (n = 110, P < 0.0001); shc-1(ok198); daf-2(e1368): 20.2 ± 0.6 d (n = 109, P < 0.0001). (F) Effect of daf-2(e1370), daf-16(m26), and age-1(hx546) on the oxidative stress sensitivity of shc-1(ok198). Animals were exposed to 200 mM and 100 mM paraquat, respectively. All data are listed as mean survival ± standard error of the mean, n = 100 per each strain. Data were combined from at least two independent experiments. (G) daf-2(e1370) increases the life span of shc-1(ok198). The assay was performed in a fer-15(b26) background at 25°C to prevent progeny. fer-15(b26): 13.0 ± 0.2 d (n = 217); shc-1(ok198); fer-15(b26): 10.7 ± 0.2 d (n = 216, P < 0.0001 vs. fer-15); fer-15(b26); daf-2(e1370): 31.8 ± 0.9 d (n = 160, P < 0.0001 vs. fer-15); shc-1(ok198); fer-15(b26); daf-2(e1370): 27.2 ± 0.6 d (n = 210, P < 0.0001 vs. fer-15). (H) The accelerated rate of lipofuscin accumulation in shc-1(ok198) is suppressed by daf-2(e1370). Quantification of the gut autofluorescence of fer-15(b26), shc-1(ok198); fer-15(b26), fer-15(b26); daf-2(e1370), and shc-1(ok198); fer-15(b26); daf-2(e1370) at day 10. Error bars represent the standard error of the mean. (***) P < 0.0001 comparing shc-1 with the shc-1; daf-2 mutant. (I) SHC-1 interacts with DAF-2 in HEK 293T cells. Membrane-bound sIg7-tagged DAF-2 (sIg.7.DAF-2) or control proteins (sIg7-tag and sIg7.control protein) were coexpressed with Flag-tagged SHC-1 (F.SHC-1) in HEK 293T cells and precipitated with Protein G Sepharose. (Top) Western blot analysis was performed with anti-Flag antibody. (Bottom) Expression levels of all proteins in the lysates and precipitates are shown.

shc-1 acts in the JNK signaling pathway for the control of life span. (A) jnk-1(gk) suppresses the shc-1(ok198) aging phenotype. Wild type: 14.2 ± 0.4 d (n = 204); shc-1(ok198): 8.4 ± 0.3 d (n = 152, P < 0.0001); jnk-1(gk7): 11.6 ± 0.2 d (n = 320, P < 0.0001); shc-1(ok198);jnk-1(gk7): 10.9 ± 0.1 d (n = 232, P < 0.0001). (B) Transgenic overexpression of jnk-1 suppresses shc-1(ok198). shc-1(ok198): 8.4 ± 0.3 d (n = 152, P < 0.0001); shc-1;lpIn1: 12.5 ± 0.3 d (n = 116, P < 0.0001); lpIn1: 14.5 ± 0.3 d (n = 123, P = 0.708). (C) shc-1 and jkk-1 act independently to control life span. Wild type: 12.0 ± 0.2 d (n = 277); shc-1(ok198): 9.5 ± 0.2 d (n = 207, P < 0.0001); jkk-1(km2): 9.5 ± 0.2 d (n = 197, P < 0.0001); shc-1(ok198);jkk-1(km2): 8.1 ± 0.2 d (n = 132, P < 0.0001). (D) Life-span analysis of shc-1(ok198) and transgenic strains expressing jkk-1. Wild type: 12.7 ± 0.2 d (n = 308); shc-1(ok198): 8.6 ± 0.2 d (n = 175, P < 0.0001); Ex[jkk-1∷gfp]: 12.5 ± 0.3 d (n = 163, P = 0.595); shc-1(ok198);Ex[jkk-1∷gfp]: 10.4 ± 0.3 d (n = 152, P < 0.0001). (E) Life-span analysis of shc-1(ok198) and mek-1(ks54). Wild type: 12.7 ± 0.2 d (n = 308); shc-1(ok198): 8.6 ± 0.2 d (n = 175, P < 0.0001); mek-1(ks54): 8.4 ± 0.2 d (n = 148, P < 0.0001); shc-1(ok198);mek-1(ks54): 8.7 ± 0.2 d (n = 139, P < 0.0001). (F) Transgenic overexpression of mek-1 suppresses shc-1 mutants. Wild type: 14.5 ± 0.3 d (n = 109); shc-1(ok198): 9.9 ± 0.3 d (n = 100, P < 0.0001); Ex[mek-1∷gfp]: 14.9 ± 0.3 d (n = 118, P = 0.467); shc-1(ok198);Ex[mek-1∷gfp]: 14.9 ± 0.4 d (n = 104, P = 0.454).

Model for the function of SHC-1 in IIS and JNK signaling. Upon DAF-2 activation, AGE-1 and PIP3 activate PDK-1 and the downstream kinase complex AKT-1/AKT-2/SGK-1. The transcription factor DAF-16 is directly inhibited and phosphorylated via activity of the IIS pathway, a function of which is induction of stress response and longevity genes. In addition, DAF-16 integrates positive signals via JNK signaling stimulating nuclear accumulation. Genetic and biochemical analyses presented here suggest that SHC-1 opposes IIS upstream of AGE-1 and DAF-2 and directly interacts with DAF-2. Furthermore, SHC-1 activates JNK signaling and interacts with MEK-1 to control life span and response against heat, oxidation, and heavy metals.

Loss of shc-1 function results in accelerated aging and increased stress sensitivity. (A) Life-span analysis of shc-1(ok198) and rescue by transgenic expression of shc-1∷gfp. shc-1(ok198): 8.9 ± 0.1 d (n = 152, P < 0.0001); wild type: 14.4 ± 0.1 d (n = 204); Ex[shc-1∷gfp]: 13.6 ± 0.5 d (n = 96, P = 0.582); shc-1(ok198); Ex[shc-1∷gfp]: 12.6 ± 0.4 d (n = 106, P = 0.0229 vs. Ex[shc-1∷gfp]). (B) shc-1(ok198) displays an increase of gut lipofuscin autofluorescence as compared with similarly aged wild-type animals at 23°C. Quantification of the wild-type and shc-1(ok198) populations’ gut autofluorescence (n = 20 per strain) at days 0, 4, and 8 of adulthood. Error bars represent the standard error of the mean. (***) P < 0.0001. (C) shc-1(ok198) is sensitive to heat stress (32°C). n = 40 per each strain and time point. (D) shc-1(ok198) is sensitive to oxidative stress (100 mM paraquat) compared with wild-type. n = 100 per each strain.

Inhibiting shc-1 function reduces DAF-16 nuclear localization in the intestinal cells. (A) Quantification of DAF-16 intestinal nuclear and cytosolic localization after 90-min heat shock in Is[daf-16∷gfp] (control animals, n = 93) and shc-1(ok198);Is[daf-16∷gfp] animals (n = 99). The graph represents multiple experiments. shc-1(ok198) reduces the number of animals with intestinal DAF-16 nuclear localization, P < 0.0001. (B) Images of intestinal cells from control animals Is[daf-16∷gfp] and shc-1(ok198); Is[daf-16∷gfp] animals displaying DAF-16 nuclear localization. Images were taken at 200× and 630× magnification.

SHC-1 acts upstream of MEK-1 to control oxidative and heavy metal stress response, and directly interacts with MEK-1. (A) shc-1, mek-1, and jnk-1 act together in oxidative stress response. jnk-1(gk7), shc-1(ok198), and double mutants display similar sensitivity to paraquat. mek-1(ks54) contributes to shc-1(ok198) oxidative stress sensitivity. A representative experiment is shown in which animals had been exposed to 100 mM paraquat. Please note the different time points for survival analysis. All data are listed as mean survival ± standard error of the mean, n = 100 per each strain. (B) shc-1(ok198) and mek-1(ks54) are sensitive to CuSO₄. The number of adults was counted after 4 d of incubation at 20°C. All data are listed as mean numbers of adults ± standard deviation of the mean, n = 400 per each strain. The graphs presented are the result of at least three representative trials. (C) SHC-1 interacts with MEK-1 in HEK 293T cells. V5-tagged MEK-1 (V5.MEK-1) or a control protein (V5-NPHP1) was coexpressed with Flag-tagged SHC-1 (F.SHC-1) in HEK 293T cells and precipitated with anti-V5 antibody. (Top) Western blot analysis was performed with anti-Flag antibody. (Bottom) Expression levels of all proteins in the lysates are shown. (D) MEK-1∷GFP and Flag-tagged SHC-1 (F.SHC-1) coimmunoprecipitate from whole-worm lysates. Total lysates were prepared from transgenic worms that express F.SHC-1 and MEK-1∷GFP or GFP as control and subjected to immunoprecipitation with anti-GFP antibody. Precipitates were probed by immunoblotting with anti-Flag (top) and anti-GFP (bottom). The expression level of F.SHC-1 in worm lysates is shown.