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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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Interaction between SEK1 and Akt1 and its role in SEK1 enzyme activity during glucose deprivation. DU-145 cells were coinfected with adenoviral vector containing His-tagged SEK1 (Ad.His-SEK1) and Ad.HA-Akt1 at an MOI of 10 (A, B, E, and F) and were transiently transfected with pFlag-JIP1-wt (wild type) or pFlag-JIP1–Thr-103A (mutant type; E). (A and B) After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h) and were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (A) or anti-His antibody (B) and were immunoblotted with anti-His or anti-HA antibody (top). The presence of His-SEK1, Akt, or phospho-Akt in the lysates was verified by immunoblotting (bottom). (C and D) DU-145 cells were exposed to glucose-free medium for 1 or 2 h and were lysed. Lysates were immunoprecipitated with anti–mouse Akt antibody. (C) Immunoprecipitates were analyzed for SEK1 binding with anti-SEK1 or anti–rabbit Akt antibody (top). The presence of SEK1 and Akt in the lysates was verified by immunoblotting (bottom). (D) Immunoprecipitates were examined for the phosphorylation of SEK1 (Ser-80) by Akt. 0.5 μg GST-SEK1 was incubated with immunoprecipitated Akt in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–phospho-SEK1 (Ser-80) antibody. The presence of GST-SEK1 or Akt in the immunoprecipitate was verified by immunoblotting with anti-SEK1 antibody or anti–rabbit Akt antibody, respectively (top). The presence of Akt, phospho-SEK1, or SEK1 in the lysates was verified by immunoblotting (bottom). (E) After 48 h of incubation, cells were exposed to glucose-free medium for 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody and were immunoblotted with anti-HA or anti-His antibody (top). The presence of HA-Akt1, Flag-JIP1 wild type, or Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting (bottom). (F) DU-145 cells were coinfected with Ad.His-SEK1 and Ad.HA-Akt1 wild type (wt) or Ad.HA-Akt1 dominant negative mutant type (DN) at an MOI of 10. After 48 h of incubation, cells were exposed to glucose-free medium for 1 or 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody. To examine the catalytic activity of SEK1, 0.5 μg GST-JNK1 was incubated with immunoprecipitated His-SEK1 in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–ACTIVE JNK antibody. The presence of GST-JNK1, His-SEK1, or HA-Akt1 in the immunoprecipitates was verified by immunoblotting (top). The presence of HA-Akt1 (wt, DN) or His-SEK1 in the lysates was verified by immunoblotting (bottom).
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fig8: Interaction between SEK1 and Akt1 and its role in SEK1 enzyme activity during glucose deprivation. DU-145 cells were coinfected with adenoviral vector containing His-tagged SEK1 (Ad.His-SEK1) and Ad.HA-Akt1 at an MOI of 10 (A, B, E, and F) and were transiently transfected with pFlag-JIP1-wt (wild type) or pFlag-JIP1–Thr-103A (mutant type; E). (A and B) After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h) and were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (A) or anti-His antibody (B) and were immunoblotted with anti-His or anti-HA antibody (top). The presence of His-SEK1, Akt, or phospho-Akt in the lysates was verified by immunoblotting (bottom). (C and D) DU-145 cells were exposed to glucose-free medium for 1 or 2 h and were lysed. Lysates were immunoprecipitated with anti–mouse Akt antibody. (C) Immunoprecipitates were analyzed for SEK1 binding with anti-SEK1 or anti–rabbit Akt antibody (top). The presence of SEK1 and Akt in the lysates was verified by immunoblotting (bottom). (D) Immunoprecipitates were examined for the phosphorylation of SEK1 (Ser-80) by Akt. 0.5 μg GST-SEK1 was incubated with immunoprecipitated Akt in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–phospho-SEK1 (Ser-80) antibody. The presence of GST-SEK1 or Akt in the immunoprecipitate was verified by immunoblotting with anti-SEK1 antibody or anti–rabbit Akt antibody, respectively (top). The presence of Akt, phospho-SEK1, or SEK1 in the lysates was verified by immunoblotting (bottom). (E) After 48 h of incubation, cells were exposed to glucose-free medium for 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody and were immunoblotted with anti-HA or anti-His antibody (top). The presence of HA-Akt1, Flag-JIP1 wild type, or Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting (bottom). (F) DU-145 cells were coinfected with Ad.His-SEK1 and Ad.HA-Akt1 wild type (wt) or Ad.HA-Akt1 dominant negative mutant type (DN) at an MOI of 10. After 48 h of incubation, cells were exposed to glucose-free medium for 1 or 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody. To examine the catalytic activity of SEK1, 0.5 μg GST-JNK1 was incubated with immunoprecipitated His-SEK1 in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–ACTIVE JNK antibody. The presence of GST-JNK1, His-SEK1, or HA-Akt1 in the immunoprecipitates was verified by immunoblotting (top). The presence of HA-Akt1 (wt, DN) or His-SEK1 in the lysates was verified by immunoblotting (bottom).

Mentions: A previous study has shown that Akt binds to SEK1 and subsequently inhibits SEK1 activity by phosphorylating mouse SEK1 on Ser-78 residue (Park et al., 2002). We postulated that the glucose deprivation–induced dissociation of Akt1 from JIP1 leads to the increased binding of Akt1 to SEK1. The interaction between Akt1 and SEK1 results in the inhibition of SEK1 enzyme activity. To test this hypothesis, we first examined Akt1–SEK1 binding during glucose deprivation. Fig. 8 (A and B) shows that binding of Akt1 to SEK1 gradually increased as a function of time during glucose deprivation without changes in the intracellular level of Akt1. We then investigated whether Akt1, which dissociates from JIP1 during glucose deprivation, is the Akt1 that interacts with SEK1. Fig. 8 (C and D) shows that endogenous Akt1 associated with endogenous SEK1 during glucose deprivation for 2 h and phosphorylated human SEK1 on Ser-80 residue. As shown previously in Fig. 4 B, Akt1 binds to JIP1 before glucose deprivation. However, Akt1 does not dissociate from the JIP1–Thr-103A mutant protein during glucose deprivation. This indicates that JIP1 is responsible for the dissociation of Akt1 from JIP1 during glucose deprivation. If it is true that the dissociation of Akt1 from JIP1 causes an increase in the interaction between Akt1 and SEK1, then that interaction should likewise be diminished by the overexpression of JIP1–Thr-103A mutant protein. Fig. 8 E shows that glucose deprivation–induced Akt1–SEK1 interaction was suppressed by overexpressing mutant-type JIP1–Thr-103A but not wild-type JIP1. These results suggest that Akt1, which has been dissociated from JIP1, interacts with SEK1. To examine whether the interaction between Akt1 and SEK1 inhibits the catalytic activity of SEK1, cells were coinfected with Ad.His-SEK1 and either Ad.HA-Akt1 wild type or Ad.HA-Akt1 mutant type (a dominant negative form carrying a point mutation that ablates the kinase activity by replacing Lys-179 residue with Met). Data from immune complex kinase assays show that GST-JNK1 was phosphorylated by His-SEK1 in cells that were deprived of glucose for 1 h (Fig. 8 F). This phosphorylation was reduced during 4 h of glucose deprivation in Ad.HA-Akt1 (wild type)–infected cells, but not in Ad.HA-Akt1–infected cells (Fig. 8 F). Altogether, these results suggest that the enzymatic activity of SEK1 is inhibited by its binding to Akt1 after several hours of glucose deprivation, resulting from activation of the negative feedback loop.


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)

Interaction between SEK1 and Akt1 and its role in SEK1 enzyme activity during glucose deprivation. DU-145 cells were coinfected with adenoviral vector containing His-tagged SEK1 (Ad.His-SEK1) and Ad.HA-Akt1 at an MOI of 10 (A, B, E, and F) and were transiently transfected with pFlag-JIP1-wt (wild type) or pFlag-JIP1–Thr-103A (mutant type; E). (A and B) After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h) and were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (A) or anti-His antibody (B) and were immunoblotted with anti-His or anti-HA antibody (top). The presence of His-SEK1, Akt, or phospho-Akt in the lysates was verified by immunoblotting (bottom). (C and D) DU-145 cells were exposed to glucose-free medium for 1 or 2 h and were lysed. Lysates were immunoprecipitated with anti–mouse Akt antibody. (C) Immunoprecipitates were analyzed for SEK1 binding with anti-SEK1 or anti–rabbit Akt antibody (top). The presence of SEK1 and Akt in the lysates was verified by immunoblotting (bottom). (D) Immunoprecipitates were examined for the phosphorylation of SEK1 (Ser-80) by Akt. 0.5 μg GST-SEK1 was incubated with immunoprecipitated Akt in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–phospho-SEK1 (Ser-80) antibody. The presence of GST-SEK1 or Akt in the immunoprecipitate was verified by immunoblotting with anti-SEK1 antibody or anti–rabbit Akt antibody, respectively (top). The presence of Akt, phospho-SEK1, or SEK1 in the lysates was verified by immunoblotting (bottom). (E) After 48 h of incubation, cells were exposed to glucose-free medium for 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody and were immunoblotted with anti-HA or anti-His antibody (top). The presence of HA-Akt1, Flag-JIP1 wild type, or Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting (bottom). (F) DU-145 cells were coinfected with Ad.His-SEK1 and Ad.HA-Akt1 wild type (wt) or Ad.HA-Akt1 dominant negative mutant type (DN) at an MOI of 10. After 48 h of incubation, cells were exposed to glucose-free medium for 1 or 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody. To examine the catalytic activity of SEK1, 0.5 μg GST-JNK1 was incubated with immunoprecipitated His-SEK1 in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–ACTIVE JNK antibody. The presence of GST-JNK1, His-SEK1, or HA-Akt1 in the immunoprecipitates was verified by immunoblotting (top). The presence of HA-Akt1 (wt, DN) or His-SEK1 in the lysates was verified by immunoblotting (bottom).
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fig8: Interaction between SEK1 and Akt1 and its role in SEK1 enzyme activity during glucose deprivation. DU-145 cells were coinfected with adenoviral vector containing His-tagged SEK1 (Ad.His-SEK1) and Ad.HA-Akt1 at an MOI of 10 (A, B, E, and F) and were transiently transfected with pFlag-JIP1-wt (wild type) or pFlag-JIP1–Thr-103A (mutant type; E). (A and B) After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h) and were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (A) or anti-His antibody (B) and were immunoblotted with anti-His or anti-HA antibody (top). The presence of His-SEK1, Akt, or phospho-Akt in the lysates was verified by immunoblotting (bottom). (C and D) DU-145 cells were exposed to glucose-free medium for 1 or 2 h and were lysed. Lysates were immunoprecipitated with anti–mouse Akt antibody. (C) Immunoprecipitates were analyzed for SEK1 binding with anti-SEK1 or anti–rabbit Akt antibody (top). The presence of SEK1 and Akt in the lysates was verified by immunoblotting (bottom). (D) Immunoprecipitates were examined for the phosphorylation of SEK1 (Ser-80) by Akt. 0.5 μg GST-SEK1 was incubated with immunoprecipitated Akt in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–phospho-SEK1 (Ser-80) antibody. The presence of GST-SEK1 or Akt in the immunoprecipitate was verified by immunoblotting with anti-SEK1 antibody or anti–rabbit Akt antibody, respectively (top). The presence of Akt, phospho-SEK1, or SEK1 in the lysates was verified by immunoblotting (bottom). (E) After 48 h of incubation, cells were exposed to glucose-free medium for 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody and were immunoblotted with anti-HA or anti-His antibody (top). The presence of HA-Akt1, Flag-JIP1 wild type, or Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting (bottom). (F) DU-145 cells were coinfected with Ad.His-SEK1 and Ad.HA-Akt1 wild type (wt) or Ad.HA-Akt1 dominant negative mutant type (DN) at an MOI of 10. After 48 h of incubation, cells were exposed to glucose-free medium for 1 or 4 h and were lysed. Lysates were immunoprecipitated with anti-His antibody. To examine the catalytic activity of SEK1, 0.5 μg GST-JNK1 was incubated with immunoprecipitated His-SEK1 in kinase buffer containing ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by immunoblotting with anti–ACTIVE JNK antibody. The presence of GST-JNK1, His-SEK1, or HA-Akt1 in the immunoprecipitates was verified by immunoblotting (top). The presence of HA-Akt1 (wt, DN) or His-SEK1 in the lysates was verified by immunoblotting (bottom).
Mentions: A previous study has shown that Akt binds to SEK1 and subsequently inhibits SEK1 activity by phosphorylating mouse SEK1 on Ser-78 residue (Park et al., 2002). We postulated that the glucose deprivation–induced dissociation of Akt1 from JIP1 leads to the increased binding of Akt1 to SEK1. The interaction between Akt1 and SEK1 results in the inhibition of SEK1 enzyme activity. To test this hypothesis, we first examined Akt1–SEK1 binding during glucose deprivation. Fig. 8 (A and B) shows that binding of Akt1 to SEK1 gradually increased as a function of time during glucose deprivation without changes in the intracellular level of Akt1. We then investigated whether Akt1, which dissociates from JIP1 during glucose deprivation, is the Akt1 that interacts with SEK1. Fig. 8 (C and D) shows that endogenous Akt1 associated with endogenous SEK1 during glucose deprivation for 2 h and phosphorylated human SEK1 on Ser-80 residue. As shown previously in Fig. 4 B, Akt1 binds to JIP1 before glucose deprivation. However, Akt1 does not dissociate from the JIP1–Thr-103A mutant protein during glucose deprivation. This indicates that JIP1 is responsible for the dissociation of Akt1 from JIP1 during glucose deprivation. If it is true that the dissociation of Akt1 from JIP1 causes an increase in the interaction between Akt1 and SEK1, then that interaction should likewise be diminished by the overexpression of JIP1–Thr-103A mutant protein. Fig. 8 E shows that glucose deprivation–induced Akt1–SEK1 interaction was suppressed by overexpressing mutant-type JIP1–Thr-103A but not wild-type JIP1. These results suggest that Akt1, which has been dissociated from JIP1, interacts with SEK1. To examine whether the interaction between Akt1 and SEK1 inhibits the catalytic activity of SEK1, cells were coinfected with Ad.His-SEK1 and either Ad.HA-Akt1 wild type or Ad.HA-Akt1 mutant type (a dominant negative form carrying a point mutation that ablates the kinase activity by replacing Lys-179 residue with Met). Data from immune complex kinase assays show that GST-JNK1 was phosphorylated by His-SEK1 in cells that were deprived of glucose for 1 h (Fig. 8 F). This phosphorylation was reduced during 4 h of glucose deprivation in Ad.HA-Akt1 (wild type)–infected cells, but not in Ad.HA-Akt1–infected cells (Fig. 8 F). Altogether, these results suggest that the enzymatic activity of SEK1 is inhibited by its binding to Akt1 after several hours of glucose deprivation, resulting from activation of the negative feedback loop.

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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Related in: MedlinePlus