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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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Effect of glucose deprivation on Akt1–JIP1/3 interaction or JIP1–JNK1/2 interaction. DU-145 cells were coinfected with Ad.HA-Akt1 and Ad.Flag-JIP1 (A) or Flag-tagged JIP3 (Ad.Flag-JIP3; B) at an MOI of 10. DU-145 cells were coinfected with Ad.Flag-JIP1 and Ad.His-JNK1 (C) or Ad.HA-JNK2 (D) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times. (A and B) Cell lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1, Flag-JIP3, phospho-Akt, or actin in the lysates was verified by immunoblotting (bottom). (C and D) Lysates were immunoprecipitated with anti-His/anti-HA antibody and were immunoblotted with anti-Flag or anti-His/anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting (bottom).
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fig3: Effect of glucose deprivation on Akt1–JIP1/3 interaction or JIP1–JNK1/2 interaction. DU-145 cells were coinfected with Ad.HA-Akt1 and Ad.Flag-JIP1 (A) or Flag-tagged JIP3 (Ad.Flag-JIP3; B) at an MOI of 10. DU-145 cells were coinfected with Ad.Flag-JIP1 and Ad.His-JNK1 (C) or Ad.HA-JNK2 (D) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times. (A and B) Cell lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1, Flag-JIP3, phospho-Akt, or actin in the lysates was verified by immunoblotting (bottom). (C and D) Lysates were immunoprecipitated with anti-His/anti-HA antibody and were immunoblotted with anti-Flag or anti-His/anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting (bottom).

Mentions: To investigate whether metabolic oxidative stress dissociates Akt1 from JIP1, DU-145 cells were infected with Ad.Flag-JIP1 and Ad.HA-Akt1. Cells were then exposed to glucose-free medium for various times, and the interaction between the two molecules was examined by immunoprecipitation. Fig. 3 A shows that Akt1 dissociated from JIP1 within 2 h during glucose deprivation. We further examined whether our findings could be generalized to other JIPs. Unlike JIP1, there was no interaction between Akt1 and JIP3 (Fig. 3 B) or JIP2 (not depicted), regardless of the glucose concentration of the medium. Next, we examined the mechanism by which Akt1 dissociates from JIP1 during glucose deprivation. We hypothesized that JNK is involved in this process. As a first step, we investigated the interaction between JIP1 and JNK1/2 during glucose deprivation. Fig. 3 (C and D) shows that the binding affinity of JNK2 to JIP1, but not that of JNK1 to JIP1, was enhanced 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)

Effect of glucose deprivation on Akt1–JIP1/3 interaction or JIP1–JNK1/2 interaction. DU-145 cells were coinfected with Ad.HA-Akt1 and Ad.Flag-JIP1 (A) or Flag-tagged JIP3 (Ad.Flag-JIP3; B) at an MOI of 10. DU-145 cells were coinfected with Ad.Flag-JIP1 and Ad.His-JNK1 (C) or Ad.HA-JNK2 (D) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times. (A and B) Cell lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1, Flag-JIP3, phospho-Akt, or actin in the lysates was verified by immunoblotting (bottom). (C and D) Lysates were immunoprecipitated with anti-His/anti-HA antibody and were immunoblotted with anti-Flag or anti-His/anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting (bottom).
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fig3: Effect of glucose deprivation on Akt1–JIP1/3 interaction or JIP1–JNK1/2 interaction. DU-145 cells were coinfected with Ad.HA-Akt1 and Ad.Flag-JIP1 (A) or Flag-tagged JIP3 (Ad.Flag-JIP3; B) at an MOI of 10. DU-145 cells were coinfected with Ad.Flag-JIP1 and Ad.His-JNK1 (C) or Ad.HA-JNK2 (D) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times. (A and B) Cell lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1, Flag-JIP3, phospho-Akt, or actin in the lysates was verified by immunoblotting (bottom). (C and D) Lysates were immunoprecipitated with anti-His/anti-HA antibody and were immunoblotted with anti-Flag or anti-His/anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting (bottom).
Mentions: To investigate whether metabolic oxidative stress dissociates Akt1 from JIP1, DU-145 cells were infected with Ad.Flag-JIP1 and Ad.HA-Akt1. Cells were then exposed to glucose-free medium for various times, and the interaction between the two molecules was examined by immunoprecipitation. Fig. 3 A shows that Akt1 dissociated from JIP1 within 2 h during glucose deprivation. We further examined whether our findings could be generalized to other JIPs. Unlike JIP1, there was no interaction between Akt1 and JIP3 (Fig. 3 B) or JIP2 (not depicted), regardless of the glucose concentration of the medium. Next, we examined the mechanism by which Akt1 dissociates from JIP1 during glucose deprivation. We hypothesized that JNK is involved in this process. As a first step, we investigated the interaction between JIP1 and JNK1/2 during glucose deprivation. Fig. 3 (C and D) shows that the binding affinity of JNK2 to JIP1, but not that of JNK1 to JIP1, was enhanced 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