The roles of shca proteins in response to oxidative stress 1

B A p-p66 S36 H2O2 (mM) 0.1 0.4 p66 0.1 0.4 p66Shc p66ShcS36A 0.5 p66 p66S36A C p-p66 S36 p66 H2O2(mM) 0.2 1.0 1.5 D p-p66 S36 p66 Time(min) 10 20 30 Figure 3.1. H2O2 treatment induces phosphorylation of Ser36 of p66shcA. (A)Migration retardation ofp66shcA and p66shcA(S36A) in response to H2O2. p66shcA and p66shcA(536A) (both myc-tagged) were overexpressed in NIH3T3 cells, which were serum starved overnight and stimulated with 0.1 or 0.4 mM H2O2 for 10 minutes. The p66shc proteins were detected by western blot using anti-myc antibodies. (B) Ser36 phosphorylation can be specifically recognized by the anti-phospho-Ser36 antibodies. NIH3T3 cells were transfected with constructs expressing p66shcA or mutant p66shcA (S36A), serum-starved, and then stimulated with H2O2 for 10 min. Normal and mutant p66shcA proteins were immunoprecipitated and analyzed by Western blot using specific antibodies. (C) Dosage studies of phosphorylation of p66shcA at Ser36 and tyrosine phosphorylation. NIH3T3 cells were treated with increasing concentrations of H2O2 for 10 min, collected, and lysed. p66shcA proteins were immunoprecipitated with anti-p66shcA antibodies. Antibodies against Ser36 phosphorylated p66shcA were used to detect Ser36 phosphorylation. (D) Cells were treated with 0.5 mM H2O2 for different periods of time. p66shcA was immunoprecipitated and Ser36 phosphorylation was determined as in A. -47- A p-p66shcS36 p66shc Time (min) 10 NIH3T3 10 JNK1-/- JNK2-/- B pErk Erk H2O2(mM) 0.25 0.5 1.0 1.5 Figure 3.2A. and B. Activation of ERK, but not JNKs, was sufficient and required for phosphorylation of p66shcA at Ser36. (A) H2O2-induced p66shcA phosphorylation at Ser36 was not reduced by JNK1/JNK2 deficiency. NIH3T3 and immortalized MEFs isolated from Jnk1-/- Jnk2-/- mice were starved and treated with 0.5 mM H2O2 for or 10 min. Endogenous p66shcA was immunoprecipitated, and its phosphorylation on Ser36 was analyzed by Western blot. (B) H2O2 treatment led to ERK activation in a dosage-dependent manner. Cells were treated with increasing concentrations of H2O2 for 10 min, and ERK activation was determined by Western blot using antibodies specifically recognizing activated ERKs. -49- C p-p66shcS36 p66shc pErk Erk H2O2 U0126(μM) - + - + - + 10 - + 15 D p-p66S36 myc(p66) MEK1Δ H2O2 - + - + p66+Vec p66+MEK1Δ Figure 3.2C. and D. Activation of ERK, but not JNKs, was sufficient and required for phosphorylation of p66shcA at Ser36. (C) Inhibition of ERK MAPKs with a specific inhibitor U0126 suppressed Ser36 phosphorylation. NIH3T3 cells were pretreated with U0126 (5, 10, 15 μM) for h before H2O2 was added. Phosphorylation of p66shcA on Ser36 was determined by Western blot analysis. (D) Coexpression of the constitutively active MEK1 (MEK1 ) led to massive phosphorylation of p66shcA on Ser36. The mutant form of MEK1 and p66shcA were used to transfect NIH3T3 cells, which were serum-starved for 20 h and stimulated with H2O2 for 10 min. p66shcA was immunoprecipitated and its Ser36 phosphorylation was determined by Western blot. -51- E GSTp66shc WB: HA(Erk) H2O2 - + - + Erk Vec p66 - + - + Erk MEK1Δ p66S36A p66 Protein loading control p66 p66S36A F WB:p-p66S36 WB: p66 IP:HA(Erk1) IB:Erk1 IP: Erk5 IB: Erk5 H2O2 - + Vec substrates p66 - + + Erk1 p66S36A - + + Erk5 p66 p66S36A Figure 3.2E. and F. Activation of ERK, but not JNKs, was sufficient and required for phosphorylation of p66shcA at Ser36. (E) ERK1 was able to phosphorylate p66shcA in vitro in a Ser36dependent manner. ERK1 and MEK were immunoprecipitated from COS7 cells treated or untreated with H2O2 and in vitro kinase assays w e r e p e r f o r m e d u s i n g GST-p66shcA and muta nt GSTp66shcA(S36A) as the substrate. (F) ERK1, but not ERK5, was able to phosphorylate p66shcA at Ser36. The experiment was carried out as in E, except that cold ATP was used and the phosphorylation was analyzed by Western blot using the specific anti-phospho-p66shcA antibodies. -52- A B IP:myc(p66) IB: HA(Erk1) IP:HA(Erk1) IB: myc(p66) IB: myc(p66) IB: HA(Erk1) WB: HA(Erk1) WB: myc(p66) H2O2 Vec - p66 p66 + H2O2 Vec - Erk + Erk - D C IP: Erk IB: p66shc IP:p66 IB: Erk IB: Erk IB: p66 IP WCL IgG - + H2O2 WCL IgG - + H2O2 Figure 3.3A.B.C. and D. Interaction between ERK1 and p66shcA. p66shcA (myc tagged) and ERK1 (HA tagged) were coexpressed in COS7 cells and co-IP experiments were carried out. (A) p66shcA and associated proteins were precipitated with anti-myc antibody, and ERK1 was detected with anti-HA. Cells transfected with DNA expressing ERK1 alone were used as control. (B) ERK1 and associated proteins were precipitated with anti-HA, and p66shcA was detected with anti-myc. Cells transfected with p66shcA alone were used as a control. (C) Co-IP of endogenous p66shcA with ERK. Cell lysates were incubated with anti-ERK1 antibodies conjugated with agarose beads overnight, washed with lysis buffer, and analyzed by Western blot using anti-p66shcA antibodies. Rabbit IgG conjugated to agarose beads was used as a control in the assays. One twentieth of the cell lysate used for IP experiment was loaded as a control. (D) CoIP of endogenous ERK with p66shcA. Cell lysates were incubated with anti-p66shcA antibodies and protein A beads, washed with lysis buffer, and analyzed by Western blot using anti-ERK antibodies. Rabbit IgG was used as a control in the assays. One twentieth of the cell lysate used for IP experiment was loaded as control. -54- E IP:Erk IB: p66 IB: HA(Erk) WB:p66 H2O2 vec + Erk1 p66WT F vec + Erk1 vec + Erk1 p66RA p66RE IP:HA(Erk) IB: shc p66 p52 p46 IB:HA(Erk) H2O2 wcl vec + Erk wcl vec p66 p52 + Erk wcl vec + Erk p46 Figure 3.3E. and F. Interaction between ERK1 and p66shcA. p66shcA (myc tagged) and ERK1 (HA tagged) were coexpressed in COS7 cells and co-IP experiments were carried out. (E) Mutations of putative docking sites of SH2 domain did not affect the interaction between ERK and p66shcA. The experiments were carried out as in B, except that mutant p66shcA proteins were used as well (RR to AA, p66RA; RR to EE, p66RE). (F) p52shcA or p46shcA did not interact with ERK1. The experiments were carried out as described in B. -56- A pAkt Akt pFOXO3a Thr32 FOXO3a H2O2(mM) 0.2 0.5 + - + 1.0 1.5 - + B pAkt Akt pFOXO3a Thr32 FOXO3a H2O2 U0126 (uM) 10 - + 15 Figure 3.4A. and B. Phosphorylation of FOXO3a at Threonine32 induced by H2O2 involved ERKs activation. (A) H2O2 treatment led to Akt activation and FOXO3a phosphorylation on Thr32. Cells were treated with increasing concentrations of H2O2 for 10 min. Akt activation and FOXO3a phosphorylation were analyzed by Western blot using anti-activated Akt antibodies and anti-phospho-FOXO3a antibodies, respectively. (B) Inhibition of ERK activation compromised H O -induced Akt activation and FOXO3a phosphorylation. NIH3T3 cells were pretreated with different concentrations of U0126 before H2O2 was added. Activation of Akt and phosphorylation of FOXO3a were analyzed by Western blot as described in Figure 3.3A. -58- C IP: p66shc IB: p-p66S36 IB: p66shc pErk Erk serum - + U0126 (μM ) - + - 10 + 20 D pAkt Akt pFOXO3a Thr32 FOXO3a Serum U0126 (μM ) - + - + 10 - + 20 Figure 3.4C. and D. Phosphorylation of FOXO3a at Thr32 induced by H2O2 involved ERKs activation. (C) Serum stimulation induced Ser36 phosphorylation required ERK activation. Cells were starved and pretreated with U0126 for h before serum was added. Endogenous p66shcA was immunoprecipitated and Ser36 phosphorylation was determined by Western blot. (D) Serum-induced activation of AKT and FOXO3a phosphorylation were not affected by inhibition of ERKs activation. The experiments were done as described in B, except that 10% serum was used to treat cells. -59- E FOXO3a TopBP1 p66Shc Time (min) 10 nuclei 30 10 30 cytosol Figure 3.4E. Phosphorylation of FOXO3a at Thr32 induced by H O involved ERKs activation. H O treatment led to translocation of FOXO3a from the nucleus to the cytoplasm. Cells were treated with H2O2 for different periods of time, harvested, and separated to the nuclear and cytoplasmic fractions. The levels of FOXO3a, TopBP1 (nucleus control), and p66shcA (cytoplasm control) were determined by western blot. Note that four fold more total proteins were loaded for the cytoplasmic fractions. -60- A p27 MnSOD β-actin H2O2(mM ) 0.1 0.2 0.4 0.5 Cont. 0.1 0.2 0.4 0.5 U0126 10 uM B p27 β-actin H2O2(mM) 0.2 0.4 Cont. 0.2 0.4 U0126 10μM Figure 3.5A. and B. H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response. (A) H2O2 treatment down-regulated expression of p27 without affecting MnSOD. Cells were treated with 0.1–0.5 mM of H2O2 for 20 h. The levels of p27 were determined by Western blot. Inhibition of ERKs was found to rescue down-regulation of p27. (B) RT-PCR experiments showed that H2O2 treatment down-regulated expression of p27 at mRNA levels. Notice that the p27 is a negative image of the gel. The experiments were done as in A. Total RNA was isolated from the cells and RT-PCR was performed using a commercial kit. -65- C p27 Actin 0.1 0.2 0.4 Shc+/+ 0.1 0.2 Shc -/- 0.4 1.2 Shc-/- Levels of p27 0.8 0.6 0.4 Shc+/+ 0.2 0.1 0.2 H2O2 (mM) 0.4 Figure 3.5C. H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response. ShcA-/- MEFs showed no down-regulation of p27 in response to H2O2. ShcA-/- and control MEFs were treated with different concentrations of H2O2 and the levels of p27 were deter mined by Western blot analysis. Bottom panel: quantitation data. -66- D p66shc actin p66 +/+ -/- p66S36A p27 actin H2O2 mM 0.1 0.2 0.3 0.4 0.1 Shc-/- +vec 0.2 0.3 0.4 Shc-/- +p66 p27 actin H2O2 mM 0.1 0.2 0.3 0.4 levels of p27 Shc-/- +p66S36A 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Shc66S36A vec Shc66 0.1 0.2 0.3 0.4 H2O2 (mM) Figure 3.5D. H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response. Ser36 of p66shcA played an important role in down-regulation of p27. ShcA-/- MEFs expressing p66shcA, p66shcA(S36A), or empty vector (top panel) were treated with different concentrations of H2O2 and the levels of p27 were determined by Western blot analysis. Bottom panel: quantitation data. -67- Cell viability E 120% WT 100% p27-/- 80% 60% 40% 20% 0% 0.2 0.3 0.4 H2O2(mM) F p27 Actin H2O2(h) 10 20 Figure 3.5E. and F. H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response. (E) p27-deficient MEFs showed an improved survival than control wild-type cells. Primary mutant and wild-type MEFs were challenged with different concentrations of H2O2 for h, and the cell survival rates were determined as described in Materials and Methods.(F) H2 O2 treatment led to rapid down-regulation of p27. Cells were treated with 0.4mM of H2O2 for different periods of time and the levels of p27 were determined by Western blot with actin as a control. -68- A pTyr p66 pTyr p52 p46 H2O2(mM) 0.25 0.5 1.0 B IP:p66 IB:pTyr p66 Time(min) 10 20 30 C IP: HA (p52) IB: pTyr IB: HA (p52) H2O2 p52 + - + Y239 /240F - + Y317F - + Y3F F i g u r e . A . B . a nd C . H O - i n du c e d Y / / phosphorylation of p46/52shcA facilitated ERK activation. (A) Dosage studies of tyrosine phosphorylation of all three ShcA isoforms in response to H2O2 treatment. Cells were treated as described in Figure 3.1D. p66shcA and p52/46shcA were precipitated with anti-p66shcA and anti-ShcA, respectively, and tyrosine phosphorylation was detected by Western blot using antiphospho-tyrosine antibodies. (B) Cells were treated with 0.5 mM H O for different periods of time; all ShcA proteins were immunoprecipitated using anti-ShcA antibodies and analyzed by Western blot using anti-phospho-tyrosine antibodies. (C) Point mutation analysis to identify the tyrosine residues that was phosphorylated in the presence of H O . p52shcA carrying mutations of Y239/240F, Y317F, or Y239/240/317F (Y3F) were expressed in NIH3T3 cells (with or without H2O2 treatment), and tyrosine phosphorylation was determined as described in A. -71- D IP:p66shc IB:pTyr p66 H2O2 - + p66 E IP: HA(Erk1) IB: pErk IB: HA(Erk1) WB: HA (p52shc) H2O2 - + p66Y3F + Vec+Erk1 F - - + p52+Erk1 - + p52Y3F +Erk1 1.0 0.8 0.6 0.4 0.2 0.0 H2O2 - + - + - + Vec+Erk1 p52+Erk1 p52Y3F+Erk1 Figure 3.6D. E. and F. H2O2-induced Y239/240/317 phosphorylation of p46/52shcA facilitated ERK activation. (D) p66shcA shared the phosphorylated tyrosine residues with p46shcA. NIH3T3 cells were transfected with p66shcA or p66shcA (Y3F) for 24 h, serum-starved for overnight, and stimulated with H2O2 for 10 min. p66shcA proteins were immunoprecipitated and the tyrosine phosphorylation was detected using anti-phospho-tyrosine antibodies. (E) ERK activation by H2O2 required tyrosine phosphorylation of p52/46shcA. NIH3T3 cells were transfected with HA-tagged ERK1 and p52shcA, p52shcA(Y3F), or vector, serum-starved for 20 h, s t i m u l a t e d w i t h . m M H O f o r mi n . E R K w a s immunoprecipitated and its activation was determined by Western blot using antibodies that specifically recognized activated ERK. The blot was stripped and reblotted with anti-HA antibodies. Western blot was carried out to check the expression of ShcA and activation of ERK. (F) Quantitation data from three repeated experiments. -73- G pErk Erk Shc H2O2(mM) 0.1 0.2 0.4 0.5 0.1 Shc+/+ 0.2 0.4 0.5 Shc-/- H IP: HA(Erk1) IB: pErk IB: HA(Erk1) WB: Myc (p66shc) H2O2 +Erk1 - + Vec 3:1 - + p66shc 3:1 - + S36A 2:1 + S36A 3:1 Figure 3.6G. and H. H2O2-induced Y239/240/317 phosphorylation of p46/52shcA facilitated ERK activation.(G) Activation of ERKs by H2O2 was diminished in ShcA-/- MEFs. Mutant and control MEFS were treated H2O2 with for different periods of time and activation of ERKs was determined by Western blot analysis. (H) Coexpression of p66shcA slightly inhibited ERK activation in a Ser36-dependent manner. NIH3T3 cells were transfected with HA-tagged ERK1 and p66shcA, p66shcA(S36A), or vector, serum-starved for 20 h, and stimulated with 0.5 mM H2O2 for 10 min. Different amounts of DNA expressing p66shcA (S36A) was used to transfect cells to test their effects on ERK activation. ERK1 was immunoprecipitated and its activation was determined by Western blot using antibodies that only recognize activated ERK. The blot was stripped and reblotted with anti-HA antibodies. Western blot was carried out to check the expression of ShcA. -75- I p-Erk Erk Myc(p66) Time(min) 7.5 10 7.5 10 p66 Vec 7.5 10 p66S36A cont p66 p66S36A 5.0 levels of p-Erk 4.0 3.0 2.0 1.0 0.0 7.5 Time (min) 10 Figure 3.6I. H2O2-induced Y239/240/317 phosphorylation of p46/52shcA facilitated ERK activation. Time course study of inhibition of ERK activation by p66shcA. COS7 cells were transfected with empty vector or constructs expressing p66shcA or p66shcA(S36A) for 24 h, serum-starved overnight, and stimulated with 0.5 mM H2O2 for different periods of time, and ERKs activation was determined by Western blot analysis. Bottom panel: quantitation data showing the activation of ERK1 and ERK2 combined. -77- A B flag flag p46 Vec GST p46 Vec Vec p52 GST GST-p52 C Vec p52 GST-p46 D IP: flag(p52) IB: HA(p46) IP: HA(p46) IB: flag(p52) IB: flag(p52) IB: HA(p46) HA(p46) H2O2 - p46+Vec - flag(p52) + p46+p52 H2O2 - - + Vec+p52 p46+p52 Figure 3.7. Interaction between p52 and p46 proteins. (A) GSTp52shcA was able to pull down p46shcA. p46shcA expressed in COS7 cells were incubated with GST-p52shcA beads overnight at 4°C. The beads were washed and p46shcA was detected on a Western blot. (B) GST-p46 was able to pull down both p52shcA and p46shcA. p52shcA and p46shcA (expressed from one mRNA) expressed in COS7 cells were incubated with GST-p46shcA beads overnight at 4°C. The beads were washed and p46shcA and p52shcA was detected by Western blot. (C) p52shcA was able to precipitate coexpressed p46shcA. COS7 cells were transfected with p52shcA (flag-tagged at C′ terminus) and p46shcA (HA tagged) and treated with H2O2 for 10 min. The cell lysates were incubated with anti-Flag antibodies and p46 was detected with anti-HA antibodies by Western blot. (D) p46shcA was able to precipitate the coexpressed p52shcA and p46shcA. The experiments were carried out as in C, except that the lysates were incubated with anti-HA antibodies and the Western blot was probed with anti-Flag antibodies. -79- A IP: myc(p66) IB:HA(p52) IB: myc(p66) WB: HA(p52) H2O2 - + - Vec+p52 + p66+p52 B IP: HA(p46) IB: myc(p66) IB: HA(p46) WB: myc(p66) H2O2 - - + Vec+p66 p46+p66 Figure 3.8A.and B. Interaction between p66shcA and p46/52shcA. (A) H2O2 promoted association between p66shcA and p52shcA. Immunoprecipitation assays were done as in C. (B) H2O2 p r o mo t ed association between p66shcA and p46shcA. Immunoprecipitation was carried out as described in Figure 3.7C. -80- C p66 IB: pTyr p52 p46 H2O2 IP: - + IgG - + - Shc + p66 IB: Shc p66 p52 p46 H2O2 + IP: IgG + Shc - + p66 Figure 3.8C. Interaction between p66shcA and p46/52shcA. Endogenous p66shcA was able to precipitate endogenous p52shcA and p46shcA in a co-IP experiment. Cells were treated with H2O2 for 10 and the cell lysates were incubated with anti-p66shcA antibodies. The Western blot was probed with anti-phospho-tyrosine and anti-ShcA antibodies, respectively. Rabbit IgG was used as a control for co-IP assays. -81- [...]... Actin 0 0 .1 0.2 0.4 0 Shc+/+ 0 .1 0.2 Shc -/- 0.4 1. 2 Shc-/- Levels of p27 1 0.8 0.6 0.4 Shc+/+ 0.2 0 0 0 .1 0.2 H2O2 (mM) 0.4 Figure 3.5C H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response ShcA- /- MEFs showed no down-regulation of p27 in response to H2O2 ShcA- /- and control MEFs were treated with different concentrations of H2O2 and the levels of p27 were deter mined... studies of tyrosine phosphorylation of all three ShcA isoforms in response to H2O2 treatment Cells were treated as described in Figure 3.1D p6 6shcA and p52/4 6shcA were precipitated with anti-p6 6shcA and anti -ShcA, respectively, and tyrosine phosphorylation was detected by Western blot using antiphospho-tyrosine antibodies (B) Cells were treated with 0.5 mM H 2 O 2 for different periods of time; all ShcA proteins. .. Ser36 of p6 6shcA played an important role in down-regulation of p27 ShcA- /- MEFs expressing p6 6shcA, p6 6shcA( S36A), or empty vector (top panel) were treated with different concentrations of H2O2 and the levels of p27 were determined by Western blot analysis Bottom panel: quantitation data -67- E Cell viability 12 0% WT 10 0% p27-/- 80% 60% 40% 20% 0% 0 0.2 0.3 0.4 H2O2(mM) F p27 Actin H2O2(h) 0 2 5 10 20... ShcA proteins were immunoprecipitated using anti -ShcA antibodies and analyzed by Western blot using anti-phospho-tyrosine antibodies (C) Point mutation analysis to identify the tyrosine residues that was phosphorylated in the presence of H 2 O 2 p5 2shcA carrying mutations of Y239/240F, Y 317 F, or Y239/240/ 317 F (Y3F) were expressed in NIH3T3 cells (with or without H2O2 treatment), and tyrosine phosphorylation... determined as described in A - 71- D IP:p66shc IB:pTyr p66 H2O2 - + p66 E IP: HA(Erk1) IB: pErk IB: HA(Erk1) WB: HA (p52shc) H2O2 - + p66Y3F + Vec+Erk1 F - - + p52+Erk1 - + p52Y3F +Erk1 1. 0 0.8 0.6 0.4 0.2 0.0 H2O2 - + - + - + Vec+Erk1 p52+Erk1 p52Y3F+Erk1 Figure 3.6D E and F H2O2-induced Y239/240/ 317 phosphorylation of p46/5 2shcA facilitated ERK activation (D) p6 6shcA shared the phosphorylated tyrosine... analysis Bottom panel: quantitation data -66- D p66shc actin p66 +/+ -/- p66S36A p27 actin H2O2 mM 0 0 .1 0.2 0.3 0.4 0 0 .1 Shc-/- +vec 0.2 0.3 0.4 Shc-/- +p66 p27 actin H2O2 mM 0 0 .1 0.2 0.3 0.4 levels of p27 Shc-/- +p66S36A 1. 6 1. 4 1. 2 1. 0 0.8 0.6 0.4 0.2 0.0 Shc66S36A vec Shc66 0 0 .1 0.2 0.3 0.4 H2O2 (mM) Figure 3.5D H2O2 treatment down-regulates expression of p27 and its role in oxidative stress response. .. 0 0 .1 0.2 0.4 0.5 0 0 .1 Shc+/+ 0.2 0.4 0.5 Shc-/- H IP: HA(Erk1) IB: pErk IB: HA(Erk1) WB: Myc (p66shc) H2O2 +Erk1 - + Vec 3 :1 - + p66shc 3 :1 - + S36A 2 :1 + S36A 3 :1 Figure 3.6G and H H2O2-induced Y239/240/ 317 phosphorylation of p46/5 2shcA facilitated ERK activation.(G) Activation of ERKs by H2O2 was diminished in ShcA- /- MEFs Mutant and control MEFS were treated H2O2 with for different periods of time... activation of ERKs was determined by Western blot analysis (H) Coexpression of p6 6shcA slightly inhibited ERK activation in a Ser36-dependent manner NIH3T3 cells were transfected with HA-tagged ERK1 and p6 6shcA, p6 6shcA( S36A), or vector, serum-starved for 20 h, and stimulated with 0.5 mM H2O2 for 10 min Different amounts of DNA expressing p6 6shcA (S36A) was used to transfect cells to test their effects... p4 6shcA NIH3T3 cells were transfected with p6 6shcA or p6 6shcA (Y3F) for 24 h, serum-starved for overnight, and stimulated with H2O2 for 10 min p6 6shcA proteins were immunoprecipitated and the tyrosine phosphorylation was detected using anti-phospho-tyrosine antibodies (E) ERK activation by H2O2 required tyrosine phosphorylation of p52/4 6shcA NIH3T3 cells were transfected with HA-tagged ERK1 and p5 2shcA, ... down p4 6shcA p4 6shcA expressed in COS7 cells were incubated with GST-p5 2shcA beads overnight at 4°C The beads were washed and p4 6shcA was detected on a Western blot (B) GST-p46 was able to pull down both p5 2shcA and p4 6shcA p5 2shcA and p4 6shcA (expressed from one mRNA) expressed in COS7 cells were incubated with GST-p4 6shcA beads overnight at 4°C The beads were washed and p4 6shcA and p5 2shcA was detected . p66S36A p66 p-p66 S36 A D p-p66 S36 Time(min) 0 5 10 20 30 p66 C p66 H 2 O 2 (mM) 0 0.2 0.5 1. 0 1. 5 p-p66 S36 Figure 3 .1. H 2 O 2 treatment induces phosphorylation of Ser36 of p6 6shcA. (A)Migration retardation ofp6 6shcA and p6 6shcA( S36A). studies of phosphorylation of p6 6shcA at Ser36 and tyrosine phosphorylation. NIH3T3 cells were treated with increasing concentrations of H 2 O 2 for 10 min, collected, and lysed. p6 6shcA proteins. sites of SH2 domain did not affect the interaction between ERK and p6 6shcA. The experiments were carried out as in B, except that mutant p6 6shcA proteins were used as well (RR to AA, p66RA; RR to