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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 Thr-183 and Tyr-185 residues of JNK2 in glucose deprivation–induced JNK2–JIP1 interaction or JIP1 phosphorylation. DU-145 cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged wild-type JNK2 (Ad.HA-JNK2-wt), Thr-183A mutant-type JNK2 (Ad.HA-JNK2–Thr-183A), or Y185F mutant-type JNK2 (Ad.HA-JNK2-Y185F) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A and B) or for 60 min (C and D) and were lysed. (A–C) Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting with anti-Flag antibody (bottom). (D) Cell lysates were immunoprecipitated with anti-HA antibody. To examine which types of JNK2 can phosphorylate JIP1, 0.5 μg GST-JIP1 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).
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fig5: Role of Thr-183 and Tyr-185 residues of JNK2 in glucose deprivation–induced JNK2–JIP1 interaction or JIP1 phosphorylation. DU-145 cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged wild-type JNK2 (Ad.HA-JNK2-wt), Thr-183A mutant-type JNK2 (Ad.HA-JNK2–Thr-183A), or Y185F mutant-type JNK2 (Ad.HA-JNK2-Y185F) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A and B) or for 60 min (C and D) and were lysed. (A–C) Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting with anti-Flag antibody (bottom). (D) Cell lysates were immunoprecipitated with anti-HA antibody. To examine which types of JNK2 can phosphorylate JIP1, 0.5 μg GST-JIP1 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).

Mentions: Fleming et al. (2000) reported that phosphorylation of Thr-183 and Tyr-185 residues is required for the full activation of JNK2. We hypothesized that phosphorylation of both residues during glucose deprivation is essential for JNK2–JIP1 interaction and the phosphorylation of JIP1. To test this possibility, Thr-183 and Tyr-185 were replaced with Ala (Thr-183A) and phenylalanine (Y185F), respectively. Fig. 5 A shows that glucose deprivation increased the interaction between JIP1 and Thr-183A mutant-type JNK2. Fig. 5 B also shows that Y185F mutant increased its binding to JIP1 during glucose deprivation. However, the total amount of JIP1 bound to the Y185F mutant-type JNK2 is much less (Fig. 5 C). Data from densitometer tracings revealed that the ratio of the relative intensity of JIP1/JNK2 of wild type is similar to that of Thr-183A. However, the ratio of the relative intensity of JIP1/JNK2 of Y185F is only 35% of the wild-type value. These data suggest that the phosphorylation of Tyr-185 residue, but not that of Thr-183 residue, facilitates the binding of JNK2 to JIP1. We further investigated whether the phosphorylation of both JNK2 residues is required for the JNK2-mediated phosphorylation of JIP1. Data from an immune complex kinase assay shows that JIP1 was phosphorylated by only wild-type JNK2 during glucose deprivation, but not by either Thr-183A or Y185F mutant-type JNK2 (Fig. 5 D). These results suggest that phosphorylation of both the Thr-183 and Tyr-185 residues is necessary for full activation of the JNK2 enzyme.


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 Thr-183 and Tyr-185 residues of JNK2 in glucose deprivation–induced JNK2–JIP1 interaction or JIP1 phosphorylation. DU-145 cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged wild-type JNK2 (Ad.HA-JNK2-wt), Thr-183A mutant-type JNK2 (Ad.HA-JNK2–Thr-183A), or Y185F mutant-type JNK2 (Ad.HA-JNK2-Y185F) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A and B) or for 60 min (C and D) and were lysed. (A–C) Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting with anti-Flag antibody (bottom). (D) Cell lysates were immunoprecipitated with anti-HA antibody. To examine which types of JNK2 can phosphorylate JIP1, 0.5 μg GST-JIP1 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).
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Related In: Results  -  Collection

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fig5: Role of Thr-183 and Tyr-185 residues of JNK2 in glucose deprivation–induced JNK2–JIP1 interaction or JIP1 phosphorylation. DU-145 cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged wild-type JNK2 (Ad.HA-JNK2-wt), Thr-183A mutant-type JNK2 (Ad.HA-JNK2–Thr-183A), or Y185F mutant-type JNK2 (Ad.HA-JNK2-Y185F) at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A and B) or for 60 min (C and D) and were lysed. (A–C) Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (top). The presence of Flag-JIP1 in the lysates was verified by immunoblotting with anti-Flag antibody (bottom). (D) Cell lysates were immunoprecipitated with anti-HA antibody. To examine which types of JNK2 can phosphorylate JIP1, 0.5 μg GST-JIP1 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).
Mentions: Fleming et al. (2000) reported that phosphorylation of Thr-183 and Tyr-185 residues is required for the full activation of JNK2. We hypothesized that phosphorylation of both residues during glucose deprivation is essential for JNK2–JIP1 interaction and the phosphorylation of JIP1. To test this possibility, Thr-183 and Tyr-185 were replaced with Ala (Thr-183A) and phenylalanine (Y185F), respectively. Fig. 5 A shows that glucose deprivation increased the interaction between JIP1 and Thr-183A mutant-type JNK2. Fig. 5 B also shows that Y185F mutant increased its binding to JIP1 during glucose deprivation. However, the total amount of JIP1 bound to the Y185F mutant-type JNK2 is much less (Fig. 5 C). Data from densitometer tracings revealed that the ratio of the relative intensity of JIP1/JNK2 of wild type is similar to that of Thr-183A. However, the ratio of the relative intensity of JIP1/JNK2 of Y185F is only 35% of the wild-type value. These data suggest that the phosphorylation of Tyr-185 residue, but not that of Thr-183 residue, facilitates the binding of JNK2 to JIP1. We further investigated whether the phosphorylation of both JNK2 residues is required for the JNK2-mediated phosphorylation of JIP1. Data from an immune complex kinase assay shows that JIP1 was phosphorylated by only wild-type JNK2 during glucose deprivation, but not by either Thr-183A or Y185F mutant-type JNK2 (Fig. 5 D). These results suggest that phosphorylation of both the Thr-183 and Tyr-185 residues is necessary for full activation of the JNK2 enzyme.

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