The roles of shca proteins in response to oxidative stress 1

Normal and mutant p66shcA proteins were immunoprecipitated and analyzed by Western blot using specific antibodies.. Endogenous p66shcA was immunoprecipitated, and its phosphorylation on

p66 p66S36A p66

p-p66 S36

A

D

p-p66

S36

Time(min) 0 5 10 20 30

p66

C

p66

H2O2(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 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 H2O2for

1 0 mi n , c o l l e c t e d , 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

B

H2O2(mM) 0 0.1 0.4 0 0.1 0.4

p66Shc p66ShcS36A

p66shc Time (min) 0 5 10 0 5 10

JNK1-/- JNK2-/-NIH3T3

p-p66shcS36

A

pErk

Erk

H2O2(mM) 0 0.25 0.5 1.0 1.5

B

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 5 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

pErk

Erk

- + - + - + - +

H2O2

C

U0126(μM) 0 5 10 15

myc(p66) p-p66S36

MEK1Δ

H2O2 - + - +

p66+Vec p66+MEK1Δ

D

p-p66shcS36

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 1 h before H2O2was 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

WB: HA(Erk)

H2O2 - + - + - + - +

GSTp66shc

Vec

p66 p66S36A Protein loading control

E

WB: p66

WB:p-p66S36

Vec Erk1 Erk5

p66 p66S36A p66 p66S36A

H2O2

substrates

IP:HA(Erk1)

IB:Erk1

F

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 Ser36-dependent manner ERK1 and MEK were immunoprecipitated from COS7 cells treated or untreated with H2O2and in vitro kinase assays were performed using p66shcA and mutant GST-p66shcA(S36A) as the substrate (F) ERK1, but not ERK5, was able

to phosphorylate p66shcA at Ser36 The experiment was carried out

IP: Erk5

IB: Erk5

Vec Erk Erk

IB: HA(Erk1)

IP:HA(Erk1) IB: myc(p66)

- +

-H2O2

B

C

IP WCL IgG - + H2O2

IP: Erk

IB: p66shc

IB: Erk

WCL IgG - + H2O2

IP:p66 IB: Erk IB: p66

D

A

IP:myc(p66)

IB: HA(Erk1)

IB: myc(p66)

WB: HA(Erk1)

Vec p66 p66

H2O2

WB: myc(p66)

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)

Co-IP 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

vec Erk1 vec Erk1 vec Erk1

H2O2 - + - + - +

IP:Erk

IB: p66

WB:p66

IB: HA(Erk)

E

IP:HA(Erk)

IB: shc

IB:HA(Erk)

wcl vec Erk wcl vec Erk wcl vec Erk

H2O2 - + - + - +

p66

p52

p46

F

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

pAkt Akt pFOXO3a

Thr32 FOXO3a

H2O2(mM) 0 0.2 0.5 1.0 1.5

- + - + - + - +

B

0 5 10 15 U0126 (uM)

H2O2

pAkt

Akt

pFOXO3a

Thr32

FOXO3a

Figure 3.4A and B Phosphorylation of FOXO3a at Threonine32 induced by H 2 O 2 involved ERKs activation (A) H2O2treatment 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 H2O2 -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

U0126 (μM ) 0 10 20

C

IP: p66shc

IB: p-p66S36

IB: p66shc

serum - + - + - +

pErk Erk

U0126 (μM ) 0 10 20

Serum - + - + - +

pAkt Akt pFOXO3a

Thr32 FOXO3a

D

Figure 3.4C and D Phosphorylation of FOXO3a at Thr32 induced by H 2 O 2 involved ERKs activation (C) Serum stimulation

induced Ser36 phosphorylation required ERK activation Cells were starved and pretreated with U0126 for 1 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

Figure 3.4E Phosphorylation of FOXO3a at Thr32 induced by

H 2 O 2 involved ERKs activation. H2O2 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

E

FOXO3a TopBP1 p66Shc Time (min) 0 10 30 0 10 30

MnSOD

U0126 10 uM Cont

β-actin

A

H2O2(mM) 0 0.2 0.4 0 0.2 0.4

B

p27 β-actin

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

Figure 3.5A and B H 2 O 2 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

0 0.1 0.2 0.4 0 0.1 0.2 0.4

p27

Actin

-/-C

Shc-/-Shc+/+

0 0.2 0.4 0.6 0.8 1 1.2

(mM)

Figure 3.5C H 2 O 2 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 determined by Western blot analysis Bottom panel: quantitation data

H2O2mM 0 0.1 0.2 0.3 0.4

p27

Shc-/- +p66S36A actin

actin

H2O2mM 0 0.1 0.2 0.3 0.4 0 0.1 0.2 0.3 0.4

p27

p66shc

+/+ p66 -/-p66S36A actin

D

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Shc66

vec Shc66S36A

Figure 3.5D H 2 O 2 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

20%

40%

60%

80%

100%

120%

H2O2(mM)

WT

p27-/-E

Figure 3.5E and F H 2 O 2 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 5 h, and the cell survival rates were determined as described in Materials and Methods.(F) H2O2 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

p27

Actin

H2O2(h) 0 2 5 10 20

F

pTyr p66

H2O2(mM) 0 0.25 0.5 1.0

pTyr

p52

p46

B

Time(min) 0 5 10 20 30

p66

IP:p66 IB:pTyr

H2O2 - + - + - + - +

IB: HA (p52)

IP: HA (p52)

IB: pTyr

C

F i g u r e 3 6 A B a n d C H 2 O 2 - i n d u c e d Y 2 3 9 / 2 4 0 / 3 1 7 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 anti-phospho-tyrosine antibodies (B) Cells were treated with 0.5 mM

H2O2 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 H2O2 p52shcA carrying mutations of Y239/240F, Y317F, or Y239/240/317F (Y3F) were

p52Y3F +Erk1

D

IP:p66shc

IB:pTyr

H2O2 - + - +

p66

p52+Erk1

H2O2 - + - + - +

Vec+Erk1

IB: HA(Erk1)

IP: HA(Erk1)

IB: pErk

E

WB: HA

(p52shc)

0.0 0.2 0.4 0.6 0.8 1.0

Vec+Erk1 p52+Erk1 p52Y3F+Erk1

H2O2

F

F i g u r e 3 6 D E a n d F H 2 O 2 - i n d u c e d Y 2 3 9 / 2 4 0 / 3 1 7 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 mu l a t e d w i t h 0 5 mM H2O2 for 10 min ERK1 was 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)

Q u a n t i t a t i o n d a t a f r o m t h r e e r e p e a t e d e x p e r i m e n t s

Shc-/-pErk

Erk

Shc

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

IP: HA(Erk1)

IB: pErk

IB: HA(Erk1)

WB: Myc

(p66shc)

H2O2 - + - + - + - +

Vec 3:1 p66shc3:1 S36A2:1

S36A 3:1 +Erk1

H

Figure 3.6G and H H 2 O 2 -induced Y239/240/317 phosphorylation

of p46/52shcA facilitated ERK activation.(G) Activation of ERKs

by H2O2was diminished in ShcA-/- MEFs Mutant and control MEFS were treated H2O2with 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 H2O2for 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

Erk

0 5 7.5 10 0 5 7.5 10 0 5 7.5 10

Myc(p66)

Time(min)

I

0.0

1.0

2.0

3.0

4.0

5.0

Time (min)

cont p66 p66S36A

Figure 3.6I H 2 O 2 -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 H2O2for 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