The roles of shca proteins in response to oxidative stress 2

p66shc and associated proteins were precipitated with anti-myc antibody and PKC δ was detected with anti-HA.. The value of co-immunoprecipitated PKC δ proteins were precipitated with ant

0 0.2 0.4 0.6 0.81 1.2 1.4 1.6

p66

p66 +

PKC δ + + + p66Shc - + +

A

IP:anti-myc(p66)

IB:anti-HA(PKC δ)

IB: anti-myc(p66)

IB:anti- myc(p66)

IP:anti-HA (PKC δ )

IB:anti-HA (PKCδ )

B

0 1 2 3 4 5 6

p66

p66Shc + + +

Fig 4.1 p66shc interacted with PKC δ and the interaction was

in COS-7 cells and coimmunoprecipitation experiments were carried out (A) p66shc and associated proteins were precipitated with anti-myc antibody and PKC δ was detected with anti-HA Right panel: quantitation data from A The value of co-immunoprecipitated PKC δ

proteins were precipitated with anti-HA and p66shc was detected

H2O2 - + - +

PKC δ+

p52Shc

PKC δ+

p46Shc

IB: anti- PKC δ

IB: anti-HA

A

p52 p46 IP: anti-HA(p46/52)

0 2 4 6 8 10

p52+

pkc -p52 +pkc

+ p46+

pkc -p46 +pkc +

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

H2O2 - + - +

PKC δ + p52Shc

PKC δ + p46Shc

IB: anti-HA(p46/52)

IB: anti- PKC δ

p52 p46 IP: anti- PKC δ

0 1 2 3 4 5

p52+

-p52 +pk

c + p46+

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

IP: anti-HA (p52)

IB: PKC δ IB: anti-HA (p52)

p52Shc - + + C

0 0.5 1 1.5 2 2.5

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

- H2O2

GFP-PKC δ

PKC δ+p52ShcA

merged myc-p52ShcA

A

Fig 4.3A Co-localization and translocation of PKC δ and ShcA

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

PKC δ p52ShcA

B

+H2O2

- H2O2

Fig 4.3B Co-localization and translocation of PKC δ and ShcA in

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

PKC δ

Erk1/2

H2O2 (min) 0 2.5 5 10 0 2.5 5 10 0 2.5 5 10

Fractions

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

P-GSTp66ShcA

Vec PKCδ GSTp66ShcA

WB: PKC δ

WB: PKCδ

WT S29A GSTp52-46

32 P-GSTp52-46

B

m/z

y 1 (R) y 2 (T) y 3 (P) y 4 (K) y y 7 (F)

6 (V) y 8 (pS) y 9 (G)

m/z

y 1 (R) y 2 (T) y 3 (P) y 4 (K) y y 7 (F)

6 (V) y 8 (pS) y 9 (G)

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)

H2O2(mM) 0 0.1 0.2 0.4 0.5 0 0.1 0.2 0.4 0.5

p-Erk1/2

Erk1/2

A

ShcA

B

p52 p46

expressing p52ShcA or p52ShcA S29A, selected for 7 days,

Inhibitor (μM) control rott 2.5 rott 5 stau 0.5 stau 1

P-Erk1/2

Erk1/2

0

1

2

3

4

5

6

7

8

cont

-cont+

Rott2

.5 μM-Rott2

.5μM+

Rott5

μM-Rott5

μM+

Stau

0.5μ

M-Stau

0.5μ

M+

Stau1

μM-Stau1

μM+

p42Erk2 p44Erk1

A

B

(A) Wild type MEFs were pre-treated with different concentration of rottlerin or staurosporin for 1 hr and the challenged with H2O2 ERK activation was analyzed with Western blot (B) Quantitation data