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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 JIP1 on Thr-103 and its role in the dissociation of JIP1 from Akt1 during glucose deprivation. (A) DU-145 cells were infected with Ad.HA-JNK2 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for 1 h and lysed. Cell lysates were immunoprecipitated with anti-HA antibody. To examine which amino acid residue of JIP1 can be phosphorylated by JNK2, 0.5 μg GST-JIP1 (wild type) or GST-JIP1–Thr-103A (mutant type) was incubated with immunoprecipitated HA-JNK2 in kinase buffer containing 100 μCi/ml γ-[32P]ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by autoradiography. The presence of GST-JIP1 and HA-JNK2 in the kinase buffer was verified by immunoblotting with anti-JIP1 antibody and anti-HA antibody, respectively (top). The presence of HA-JNK2 in the lysates was verified by immunoblotting with anti-HA antibody (bottom). (B) DU-145 cells were transfected with pFlag-JIP1–Thr-103A and infected with Ad.HA-Akt1. After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h). Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting with anti-Flag antibody (bottom).
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fig4: Phosphorylation of JIP1 on Thr-103 and its role in the dissociation of JIP1 from Akt1 during glucose deprivation. (A) DU-145 cells were infected with Ad.HA-JNK2 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for 1 h and lysed. Cell lysates were immunoprecipitated with anti-HA antibody. To examine which amino acid residue of JIP1 can be phosphorylated by JNK2, 0.5 μg GST-JIP1 (wild type) or GST-JIP1–Thr-103A (mutant type) was incubated with immunoprecipitated HA-JNK2 in kinase buffer containing 100 μCi/ml γ-[32P]ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by autoradiography. The presence of GST-JIP1 and HA-JNK2 in the kinase buffer was verified by immunoblotting with anti-JIP1 antibody and anti-HA antibody, respectively (top). The presence of HA-JNK2 in the lysates was verified by immunoblotting with anti-HA antibody (bottom). (B) DU-145 cells were transfected with pFlag-JIP1–Thr-103A and infected with Ad.HA-Akt1. After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h). Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting with anti-Flag antibody (bottom).

Mentions: Previously, Nihalani et al. (2003) reported that JNK is involved in the Thr-103 phosphorylation of JIP1, leading to the dissociation of dual zipper-bearing kinase (DLK) from JIP1. JNK recruitment to JIP1 is related to the decreased affinity of JIP1 and DLK (Nihalani et al., 2001). We postulated that metabolic oxidative stress-induced JNK2 activation promotes the interaction between JNK2 and JIP1 and leads to the phosphorylation of JIP1 on Thr-103, thereby causing the dissociation of Akt1 from JIP1. To examine this possibility, we used site-directed mutagenesis to create a point mutation at residue Thr-103 (Thr → Ala) of JIP1 and measured the effect on the dissociation of Akt1 from JIP1 during glucose deprivation. Data from immune complex kinase assays show that the glucose deprivation–induced phosphorylation of JIP1 by JNK2 was diminished in Thr-103A mutant-type JIP1 (Fig. 4 A). Unlike wild-type JIP1, the interaction between Akt1 and Thr-103A mutant-type JIP1 was maintained during glucose deprivation (Figs. 3 A and 4 B). These data suggest that JNK2 binds to JIP1 and phosphorylates JIP1 on the Thr-103 residue during glucose deprivation. The phosphorylation of JIP1 on Thr-103 leads to the dissociation of Akt1 from 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 JIP1 on Thr-103 and its role in the dissociation of JIP1 from Akt1 during glucose deprivation. (A) DU-145 cells were infected with Ad.HA-JNK2 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for 1 h and lysed. Cell lysates were immunoprecipitated with anti-HA antibody. To examine which amino acid residue of JIP1 can be phosphorylated by JNK2, 0.5 μg GST-JIP1 (wild type) or GST-JIP1–Thr-103A (mutant type) was incubated with immunoprecipitated HA-JNK2 in kinase buffer containing 100 μCi/ml γ-[32P]ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by autoradiography. The presence of GST-JIP1 and HA-JNK2 in the kinase buffer was verified by immunoblotting with anti-JIP1 antibody and anti-HA antibody, respectively (top). The presence of HA-JNK2 in the lysates was verified by immunoblotting with anti-HA antibody (bottom). (B) DU-145 cells were transfected with pFlag-JIP1–Thr-103A and infected with Ad.HA-Akt1. After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h). Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting with anti-Flag antibody (bottom).
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fig4: Phosphorylation of JIP1 on Thr-103 and its role in the dissociation of JIP1 from Akt1 during glucose deprivation. (A) DU-145 cells were infected with Ad.HA-JNK2 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for 1 h and lysed. Cell lysates were immunoprecipitated with anti-HA antibody. To examine which amino acid residue of JIP1 can be phosphorylated by JNK2, 0.5 μg GST-JIP1 (wild type) or GST-JIP1–Thr-103A (mutant type) was incubated with immunoprecipitated HA-JNK2 in kinase buffer containing 100 μCi/ml γ-[32P]ATP at 30°C for 1 h. Phosphorylated proteins were resolved by SDS-PAGE and were analyzed by autoradiography. The presence of GST-JIP1 and HA-JNK2 in the kinase buffer was verified by immunoblotting with anti-JIP1 antibody and anti-HA antibody, respectively (top). The presence of HA-JNK2 in the lysates was verified by immunoblotting with anti-HA antibody (bottom). (B) DU-145 cells were transfected with pFlag-JIP1–Thr-103A and infected with Ad.HA-Akt1. After 48 h of incubation, cells were exposed to glucose-free medium for various times (1–4 h). Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1–Thr-103A in the lysates was verified by immunoblotting with anti-Flag antibody (bottom).
Mentions: Previously, Nihalani et al. (2003) reported that JNK is involved in the Thr-103 phosphorylation of JIP1, leading to the dissociation of dual zipper-bearing kinase (DLK) from JIP1. JNK recruitment to JIP1 is related to the decreased affinity of JIP1 and DLK (Nihalani et al., 2001). We postulated that metabolic oxidative stress-induced JNK2 activation promotes the interaction between JNK2 and JIP1 and leads to the phosphorylation of JIP1 on Thr-103, thereby causing the dissociation of Akt1 from JIP1. To examine this possibility, we used site-directed mutagenesis to create a point mutation at residue Thr-103 (Thr → Ala) of JIP1 and measured the effect on the dissociation of Akt1 from JIP1 during glucose deprivation. Data from immune complex kinase assays show that the glucose deprivation–induced phosphorylation of JIP1 by JNK2 was diminished in Thr-103A mutant-type JIP1 (Fig. 4 A). Unlike wild-type JIP1, the interaction between Akt1 and Thr-103A mutant-type JIP1 was maintained during glucose deprivation (Figs. 3 A and 4 B). These data suggest that JNK2 binds to JIP1 and phosphorylates JIP1 on the Thr-103 residue during glucose deprivation. The phosphorylation of JIP1 on Thr-103 leads to the dissociation of Akt1 from 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