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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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Phosphorylation of Akt on Thr-308 and Ser-473 and its role in the association of Akt1 with JIP1. (A and B) DU-145 cells were infected with adenoviral vector containing HA-tagged Akt1 (Ad.HA-Akt1) and/or Ad.Flag-JIP1 at an MOI of 10. After 48 h of infection, cells were lysed. Lysates were immunoprecipitated with anti-HA antibody or anti-Flag antibody. Immunoprecipitated proteins and lysates were separated by SDS-PAGE and were immunoblotted with anti–phospho–Thr-308–Akt, anti–phospho–Ser-473–Akt, anti-Flag, anti-HA, or antiactin antibody. (C and D) DU-145 cells were transfected with pHA-Akt1 (wild type), pHA-Akt1 (Thr-308A), pHA-Akt1 (Ser-473A), or pHA-Akt1 (Thr-308A + Ser-473A) plasmids and were infected with Ad.Flag-JIP1 at an MOI of 10. After 48 h of incubation, cells were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (C) or anti-Flag antibody (D) and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 or HA-Akt1 in the lysates was verified by immunoblotting with anti-Flag antibody or anti-HA antibody, respectively (bottom).
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fig2: Phosphorylation of Akt on Thr-308 and Ser-473 and its role in the association of Akt1 with JIP1. (A and B) DU-145 cells were infected with adenoviral vector containing HA-tagged Akt1 (Ad.HA-Akt1) and/or Ad.Flag-JIP1 at an MOI of 10. After 48 h of infection, cells were lysed. Lysates were immunoprecipitated with anti-HA antibody or anti-Flag antibody. Immunoprecipitated proteins and lysates were separated by SDS-PAGE and were immunoblotted with anti–phospho–Thr-308–Akt, anti–phospho–Ser-473–Akt, anti-Flag, anti-HA, or antiactin antibody. (C and D) DU-145 cells were transfected with pHA-Akt1 (wild type), pHA-Akt1 (Thr-308A), pHA-Akt1 (Ser-473A), or pHA-Akt1 (Thr-308A + Ser-473A) plasmids and were infected with Ad.Flag-JIP1 at an MOI of 10. After 48 h of incubation, cells were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (C) or anti-Flag antibody (D) and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 or HA-Akt1 in the lysates was verified by immunoblotting with anti-Flag antibody or anti-HA antibody, respectively (bottom).

Mentions: It is well known that Akt1 is activated by phosphoinositide-dependent kinase 1 through phosphorylation at the Thr-308 and Ser-473 residues (Alessi et al., 1997). Fig. 2 (A and B) shows that Akt1 phosphorylated on Thr-308 and that Ser-473 binds to JIP1. To determine whether both residues play an important role in the interaction between Akt1 and JIP1, site-directed mutagenesis was used to create point mutations in one or both residues, converting them to Ala residues (Thr-308A, Ser-473A, and Thr-308A + Ser-473A). Fig. 2 (C and D) clearly demonstrates that there is no difference between wild-type Akt1 and mutant-type Akt1s in terms of binding to JIP1. These results indicate that the phosphorylation of these residues is not essential for the binding of Akt1 to JIP1.


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)

Phosphorylation of Akt on Thr-308 and Ser-473 and its role in the association of Akt1 with JIP1. (A and B) DU-145 cells were infected with adenoviral vector containing HA-tagged Akt1 (Ad.HA-Akt1) and/or Ad.Flag-JIP1 at an MOI of 10. After 48 h of infection, cells were lysed. Lysates were immunoprecipitated with anti-HA antibody or anti-Flag antibody. Immunoprecipitated proteins and lysates were separated by SDS-PAGE and were immunoblotted with anti–phospho–Thr-308–Akt, anti–phospho–Ser-473–Akt, anti-Flag, anti-HA, or antiactin antibody. (C and D) DU-145 cells were transfected with pHA-Akt1 (wild type), pHA-Akt1 (Thr-308A), pHA-Akt1 (Ser-473A), or pHA-Akt1 (Thr-308A + Ser-473A) plasmids and were infected with Ad.Flag-JIP1 at an MOI of 10. After 48 h of incubation, cells were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (C) or anti-Flag antibody (D) and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 or HA-Akt1 in the lysates was verified by immunoblotting with anti-Flag antibody or anti-HA antibody, respectively (bottom).
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Related In: Results  -  Collection

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fig2: Phosphorylation of Akt on Thr-308 and Ser-473 and its role in the association of Akt1 with JIP1. (A and B) DU-145 cells were infected with adenoviral vector containing HA-tagged Akt1 (Ad.HA-Akt1) and/or Ad.Flag-JIP1 at an MOI of 10. After 48 h of infection, cells were lysed. Lysates were immunoprecipitated with anti-HA antibody or anti-Flag antibody. Immunoprecipitated proteins and lysates were separated by SDS-PAGE and were immunoblotted with anti–phospho–Thr-308–Akt, anti–phospho–Ser-473–Akt, anti-Flag, anti-HA, or antiactin antibody. (C and D) DU-145 cells were transfected with pHA-Akt1 (wild type), pHA-Akt1 (Thr-308A), pHA-Akt1 (Ser-473A), or pHA-Akt1 (Thr-308A + Ser-473A) plasmids and were infected with Ad.Flag-JIP1 at an MOI of 10. After 48 h of incubation, cells were lysed. Cell lysates were immunoprecipitated with anti-HA antibody (C) or anti-Flag antibody (D) and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 or HA-Akt1 in the lysates was verified by immunoblotting with anti-Flag antibody or anti-HA antibody, respectively (bottom).
Mentions: It is well known that Akt1 is activated by phosphoinositide-dependent kinase 1 through phosphorylation at the Thr-308 and Ser-473 residues (Alessi et al., 1997). Fig. 2 (A and B) shows that Akt1 phosphorylated on Thr-308 and that Ser-473 binds to JIP1. To determine whether both residues play an important role in the interaction between Akt1 and JIP1, site-directed mutagenesis was used to create point mutations in one or both residues, converting them to Ala residues (Thr-308A, Ser-473A, and Thr-308A + Ser-473A). Fig. 2 (C and D) clearly demonstrates that there is no difference between wild-type Akt1 and mutant-type Akt1s in terms of binding to JIP1. These results indicate that the phosphorylation of these residues is not essential for the binding of Akt1 to JIP1.

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