A B Fold IP:anti-HA (PKC δ ) IB:anti- myc(p66) IB:anti-HA (PKCδ ) PKC δ p66Shc H2O2 + - + + - + + + pk c +p66 + + + + 1.6 1.4 1.2 0.8 0.6 0.4 0.2 pkc+p66 + + + - pk c +p66 - + - pkc+p66 - PKC δ p66Shc H2O2 Fold IP:anti-myc(p66) IB:anti-HA(PKC δ) IB: anti-myc(p66) Fig. 4.1. p66shc interacted with PKC δ and the interaction was enhanced by H2O2 treatment. p66shc and PKC δ were co-expressed in COS-7 cells and coimmunoprecipitation experiments were carried out. (A). p66shc and associated proteins were precipitated with antimyc antibody and PKC δ was detected with anti-HA. Right panel: quantitation data from A. The value of co-immunoprecipitated PKC δ in the absence of H2O2 was set at 1.( B). PKC δ and associated proteins were precipitated with anti-HA and p66shc was detected with anti-Myc. Right panel: quantitation data from B. The value of co-immunoprecipitated p66Shc in the absence of H2O2 was set at 1. “-”: no H2O2; “+” : H2O2. -92- A IP: anti-HA(p46/52) IB: anti- PKC δ IB: anti-HA H2O2 - + PKC δ+ p52Shc - + p52 p46 PKC δ+ p46Shc Fold 10 p5 2+ p5 pkc 2+ pk p4 c 6+ + p4 pkc 6+ pk c + Fig. 4.2A. Association between p52ShcA and p46ShcA with PKC δ. p52ShcA or p46ShcA and associated proteins were precipitated with anti-ShcA antibodies and PKC δ was detected with an anti-PKC δ antibody. Bottom panel: quantitation data from A. The value of coimmunoprecipitated PKC δ in the absence of H2O2 was set at 1. -94- B p52 p46 IP: anti- PKC δ IB: anti-HA(p46/52) IB: anti- PKC δ H2O2 - + + PKC δ + p46Shc p5 2+ p5 pkc 2+ p p4 kc 6+ + p p4 kc 6+ pk c+ Fold PKC δ + p52Shc - Fig. 4.2B Association between p52ShcA and p46ShcA with PKC δ. PKC δ and associated proteins were precipitated with anti-PKC δ antibodies and p46shc was detected with anti-ShcA antibodies. Note in A and B, p52 construct expressed both p52 and p46 Shc due to the use of two start codons. Bottom panel: quantitation data from B. The value of co-immunoprecipitated p52Shc or p46Shc in the absence of H2O2was set at 1. -95- C IP: anti-HA (p52) IB: PKC δ H2O2 + - + p52Shc - + + p52+pkc - p52+pkc + IB: anti-HA (p52) 2.5 Fold 1.5 0.5 Fig. 4.2C Association between p52ShcA and p46ShcA with PKC δ. The experiment was done as Fig. 2A, except that this p52ShcA construct did not express p46 due to a point mutation. Bottom panel: quantitation data from C. The value of co-immunoprecipitated PKC δ in the absence of H2O2 was set at 1. . “-” : no H2O2; “+” : H2O2. -96- A PKC δ+p52ShcA - H2O2 +H2O2 GFP-PKC δ myc-p52ShcA merged Fig. 4.3A. Co-localization and translocation of PKC δ and ShcA in response to H2O2 treatment. COS7 cells were transfected with PKC δ-GFP, p52shc, or both for 48 hours and then treated with H2O2. Cells were then fixed, stained for p52ShcA with anti-myc antibody and secondary anti-mouse antibodies conjugated with Texas-Red, and visualized under a confocal microscope. Cells expressing both PKCδ-GFP and p66shc. -98- B PKC δ p52ShcA - H2O2 +H2O2 GFP-PKC δ myc-p52ShcA Fig. 4.3B. Co-localization and translocation of PKC δ and ShcA in response to H2O2 treatment. COS7 cells were transfected with PKC δ-GFP, p52shc, or both for 48 hours and then treated with H2O2. Cells were then fixed, stained for p52ShcA with anti-myc antibody and secondary anti-mouse antibodies conjugated with Texas-Red, and visualized under a confocal microscope. Cells expressing either PKC δ or p66shc. -99- ShcA PKC δ Erk1/2 H2O2 (min) Fractions 2.5 10 total lysate 2.5 10 particulate 2.5 10 cytosol Fig. 4.4. Cell fractionation studies of PKC δ and ShcA. NIH3T3 cells were treated with H2O2 for 2.5, 5, 10 mins, collected and separated into cytosolic and particulate fractions. The levels of PKC δ and ShcA were analyzed with Western blot and compared with those from total lysates. ERK1/2 was used as a control. -100- C A 32 PGSTp66ShcA 32 PGSTp52-46 WB: PKCδ WB: PKC δ GSTp52-46 GSTp66ShcA WT S29A Vec PKCδ B HGpSFVNKPTR (m/z 611.7891, 2+) y2(T) y3(P) y4(K) y6(V) y7(F) y8(pS) y9(G) Intensity (counts) y1(R) m/z Fig. 4.5. PKC δ phosphorylates ShcA. (A). In vitro kinase assay shows that GST-p66ShcA was phosphorylated by PKC δ. (B). Mass spectrometric analysis of phosphorylation sites on ShcA. Tandem mass spectrum showing the sequence of a peptide of m/z 611.7891 which was eluted from the reversed phase column at 37.15 min. The y-ion series shows the sequence that was determined and that a serine residue at position three from the Nterminus was phosphorylated. The phosphorylation was confirmed by the loss of 98 amu (as -H3PO4) from the serine residue which occurs during fragmentation. (C). In vitro kinase assay shows that p52ShcA was phosphorylated by PKC δ at Ser29. GST-p52Shc (6KD fragment from N’terminus that distinguishes p52 from p46) and the mutant version (Ser29A) were used as substrates for in vitro kinase assay as described in Fig. 4.5A. -102- A p-Erk1/2 Erk1/2 H2O2(mM) 0.1 0.2 0.4 0.5 pMSCVp52ShcWT 0.1 0.2 0.4 0.5 pMSCVp52ShcS29A B p52 p46 pMSCVp52S29A pMSCVp52WT ShcA-/- ShcA Fig. 4.6. Ser29Ala mutation diminishes H O induced ERK activation. (A). ShcA-/- MEFs were infected with retroviruses expressing p52ShcA or p52ShcA S29A, selected for days, challenged with different concentrations of H2O2. ERK activation was analyzed with Western blot. (B). Expression of p52ShcA or p52ShcA S29A in retroviral infected MEFs. -104- A P-Erk1/2 Erk1/2 H2O2 - + Inhibitor (μM) control - + rott 2.5 - + rott - + stau 0.5 - + stau B p42Erk2 p44Erk1 co nt co R ot nt+ t2 .5 R ot μM t2 .5 μM R ot + t5 μM R ot t5 St μ au M+ St .5μ M au 0. 5μ St M+ au St μM au 1μ M + Levels of activated ERKs Fig. 4.7. PKC inhibitors diminish H2O2 induced ERK activation. (A). Wild type MEFs were pre-treated with different concentration of rottlerin or staurosporin for hr and the challenged with H2O2. ERK activation was analyzed with Western blot. (B). Quantitation data from A. The basal level of activated ERK2 was set at 1. Rott: Rottlerin; Stau: staurosporin. -105- . δ in the absence of H 2 O 2 was set at 1. . “-” : no H 2 O 2 ; “+” : H 2 O 2 . -96- +H 2 O 2 -H 2 O 2 GFP-PKC δ PKC δ+p5 2ShcA mergedmyc-p5 2ShcA A Fig. 4.3A. Co-localization and translocation of. described in Fig. 4.5A. -1 02- H 2 O 2 (mM) 0 0.1 0 .2 0.4 0.5 0 0.1 0 .2 0.4 0.5 p-Erk1 /2 Erk1 /2 pMSCVp52ShcWT pMSCVp52ShcS29A A ShcA pMSCVp52WT ShcA- /- pMSCVp52S29A B p 52 p46 Fig. 4.6. Ser29Ala mutation. expressing both PKCδ-GFP and p66shc. -98- PKC δ p5 2ShcA GFP-PKC δ myc-p5 2ShcA B +H 2 O 2 -H 2 O 2 Fig. 4.3B. Co-localization and translocation of PKC δ and ShcA in response to H 2 O 2 treatment.