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Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop.

Song JJ, Lee YJ - J. Cell Biol. (2005)

Bottom Line: We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway.Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation.Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

ABSTRACT
We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

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Role of Akt1 in glucose deprivation–induced JNK activation and morphological damage. (A and B) DU-145 cells were transfected with Akt1 siRNA or mock siRNA and were incubated for 36 h. (A) Akt1 protein expression was assessed by immunoblotting with anti-Akt1 antibody. (B) Cells were exposed to glucose-free medium for various times (10–120 min). Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, or antiactin antibody. (C and D) DU-145 cells were transfected with Akt1 or mock siRNA. After 24 of incubation, cells were infected with Ad.EGFP or Ad.HA-Akt1 at an MOI of 10. After 24 h of infection, cells were exposed to glucose-free medium for 60 min (C) or for 24 h (D). (C) Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, anti-Akt1, or antiactin antibody. (D) Morphology was evaluated with a phase-contrast microscope.
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fig10: Role of Akt1 in glucose deprivation–induced JNK activation and morphological damage. (A and B) DU-145 cells were transfected with Akt1 siRNA or mock siRNA and were incubated for 36 h. (A) Akt1 protein expression was assessed by immunoblotting with anti-Akt1 antibody. (B) Cells were exposed to glucose-free medium for various times (10–120 min). Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, or antiactin antibody. (C and D) DU-145 cells were transfected with Akt1 or mock siRNA. After 24 of incubation, cells were infected with Ad.EGFP or Ad.HA-Akt1 at an MOI of 10. After 24 h of infection, cells were exposed to glucose-free medium for 60 min (C) or for 24 h (D). (C) Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, anti-Akt1, or antiactin antibody. (D) Morphology was evaluated with a phase-contrast microscope.

Mentions: Our studies have revealed that Akt1 acts as a negative regulator for the ASK1–SEK1–JNK pathway during glucose deprivation. To confirm our observations, cells were transfected with Akt1 or mock siRNA. Fig. 10 A shows that the expression of Akt1 was effectively inhibited by siAkt1. Glucose deprivation–induced JNK activation (Fig. 10, B and C) and cytotoxicity (Fig. 10 D) were promoted in siAkt1-transfected cells. To exclude off-target effects of the siAkt construct, siAkt1-transfected cells were infected with Ad.HA-Akt1. Fig. 10 (C and D) shows that the overexpression of Akt1 in siAkt1-transfected cells suppressed glucose deprivation–induced JNK activation and cytotoxicity. These results suggest that Akt1 acts as a negative regulator for the ASK1–SEK1–JNK pathway during glucose deprivation.


Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop.

Song JJ, Lee YJ - J. Cell Biol. (2005)

Role of Akt1 in glucose deprivation–induced JNK activation and morphological damage. (A and B) DU-145 cells were transfected with Akt1 siRNA or mock siRNA and were incubated for 36 h. (A) Akt1 protein expression was assessed by immunoblotting with anti-Akt1 antibody. (B) Cells were exposed to glucose-free medium for various times (10–120 min). Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, or antiactin antibody. (C and D) DU-145 cells were transfected with Akt1 or mock siRNA. After 24 of incubation, cells were infected with Ad.EGFP or Ad.HA-Akt1 at an MOI of 10. After 24 h of infection, cells were exposed to glucose-free medium for 60 min (C) or for 24 h (D). (C) Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, anti-Akt1, or antiactin antibody. (D) Morphology was evaluated with a phase-contrast microscope.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171369&req=5

fig10: Role of Akt1 in glucose deprivation–induced JNK activation and morphological damage. (A and B) DU-145 cells were transfected with Akt1 siRNA or mock siRNA and were incubated for 36 h. (A) Akt1 protein expression was assessed by immunoblotting with anti-Akt1 antibody. (B) Cells were exposed to glucose-free medium for various times (10–120 min). Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, or antiactin antibody. (C and D) DU-145 cells were transfected with Akt1 or mock siRNA. After 24 of incubation, cells were infected with Ad.EGFP or Ad.HA-Akt1 at an MOI of 10. After 24 h of infection, cells were exposed to glucose-free medium for 60 min (C) or for 24 h (D). (C) Cell lysates were immunoblotted with anti–ACTIVE JNK, anti-JNK2, anti-Akt1, or antiactin antibody. (D) Morphology was evaluated with a phase-contrast microscope.
Mentions: Our studies have revealed that Akt1 acts as a negative regulator for the ASK1–SEK1–JNK pathway during glucose deprivation. To confirm our observations, cells were transfected with Akt1 or mock siRNA. Fig. 10 A shows that the expression of Akt1 was effectively inhibited by siAkt1. Glucose deprivation–induced JNK activation (Fig. 10, B and C) and cytotoxicity (Fig. 10 D) were promoted in siAkt1-transfected cells. To exclude off-target effects of the siAkt construct, siAkt1-transfected cells were infected with Ad.HA-Akt1. Fig. 10 (C and D) shows that the overexpression of Akt1 in siAkt1-transfected cells suppressed glucose deprivation–induced JNK activation and cytotoxicity. These results suggest that Akt1 acts as a negative regulator for the ASK1–SEK1–JNK pathway during glucose deprivation.

Bottom Line: We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway.Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation.Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

ABSTRACT
We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.

Show MeSH
Related in: MedlinePlus