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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 JIP1 in Akt activity in DU-145 cells. (A) Cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed, and lysates were immunoprecipitated (IP) with anti–mouse Akt1 antibody. Immunoprecipitates were analyzed for the interaction of JIP1 with Akt1 (with anti-JIP1 antibody) and Akt1 catalytic activity in vitro using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti-JIP1, anti-phospho–Ser-473–Akt1, anti-Akt1, or antiactin antibody. (B) Immunoblot of JIP1 expression in control vector transfected (pSilencer) or pSilencer-siJIP1 stably transfected (siJIP1#1–3) single cell clones from DU-145 cells. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and were immunoblotted with anti-JIP1 antibody. (C) Control plasmid or pSilencer-siJIP1 stably transfected siJIP1#2 cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt1 antibody. Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti–phospho–Ser-473–Akt1, anti-JIP1, or antiactin antibody. (D) Cells were infected with adenoviral vector containing Flag-tagged JIP1 cDNA (Ad.Flag-JIP1) at various multiplicity of infections (MOIs; 2–50). After 48 h of infection, cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt antibody. Immunoprecipitates were analyzed for Akt catalytic activity and JIP1 binding using immunoprecipitated Akt as described in Fig. 1 A (top). The presence of JIP1 or actin in the lysates was verified by immunoblotting (bottom). (E and F) Cells were infected with Ad.EGFP and/or Ad.Flag-JIP1 at various MOIs (10–200). After 48 h of infection, morphology was evaluated with a phase-contrast microscope (E), or cell lysates were immunoblotted with anti-PARP, anti–caspase-9, anti-JIP1, or antiactin antibody (F).
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fig1: Role of JIP1 in Akt activity in DU-145 cells. (A) Cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed, and lysates were immunoprecipitated (IP) with anti–mouse Akt1 antibody. Immunoprecipitates were analyzed for the interaction of JIP1 with Akt1 (with anti-JIP1 antibody) and Akt1 catalytic activity in vitro using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti-JIP1, anti-phospho–Ser-473–Akt1, anti-Akt1, or antiactin antibody. (B) Immunoblot of JIP1 expression in control vector transfected (pSilencer) or pSilencer-siJIP1 stably transfected (siJIP1#1–3) single cell clones from DU-145 cells. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and were immunoblotted with anti-JIP1 antibody. (C) Control plasmid or pSilencer-siJIP1 stably transfected siJIP1#2 cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt1 antibody. Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti–phospho–Ser-473–Akt1, anti-JIP1, or antiactin antibody. (D) Cells were infected with adenoviral vector containing Flag-tagged JIP1 cDNA (Ad.Flag-JIP1) at various multiplicity of infections (MOIs; 2–50). After 48 h of infection, cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt antibody. Immunoprecipitates were analyzed for Akt catalytic activity and JIP1 binding using immunoprecipitated Akt as described in Fig. 1 A (top). The presence of JIP1 or actin in the lysates was verified by immunoblotting (bottom). (E and F) Cells were infected with Ad.EGFP and/or Ad.Flag-JIP1 at various MOIs (10–200). After 48 h of infection, morphology was evaluated with a phase-contrast microscope (E), or cell lysates were immunoblotted with anti-PARP, anti–caspase-9, anti-JIP1, or antiactin antibody (F).

Mentions: A previous study has shown that Akt1 binds to JIP1, which is a JNK pathway scaffold protein (Kim et al., 2002). We hypothesized that JIP1 acts as a negative regulator of Akt1 and that JIP1–Akt1 interaction results in the inhibition of Akt1 catalytic activity. Metabolic oxidative stress may dissociate Akt1 from JIP1, thereby restoring Akt1 enzyme activity. To test the hypothesis, which was the first step in this study, we examined whether endogenous JIP1 associates with endogenous Akt1 and inhibits the enzymatic activity of Akt1 and whether metabolic oxidative stress dissociates Akt1 from JIP1, thereby restoring Akt1 enzyme activity. DU-145 cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed and immunoprecipitated with anti-Akt1 antibody followed by immunoblotting with anti-JIP1 antibody, or Akt1 enzyme activity was measured by using an immune complex kinase assay. Fig. 1 A shows that endogenous Akt1 interacted with endogenous JIP1 (lane 2). However, Akt1 dissociated from JIP1 within 2 h during glucose deprivation (Fig. 1 A, lane 6). Fig. 1 A also shows that Akt1 phosphorylated an Akt-specific substrate, Bad (lane 6). These results suggest that Akt1 interacts with JIP1 and that Akt1 catalytic activity is inhibited. The dissociation of Akt1 from JIP1 restores Akt1 enzyme activity, and the role of JIP1 in Akt1 catalytic activity was further examined by the knockdown of JIP1 expression. Unlike pSilencer control plasmid–transfected cells, pSilencer–short interference (si)JIP1 stably transfected siJIP1#2 cells contained a low level of JIP1 (Fig. 1 B) and promoted Akt1 enzyme activity (Fig. 1 C). In contrast, when JIP1 was overexpressed, more JIP1 associated with Akt1, and the phosphorylation of Bad on Ser-136 was inhibited (Fig. 1 D, lanes 4–6). The inhibition of Bad phosphorylation was dependent on the level of JIP1 expression (Fig. 1 D). These results suggest that the interaction between Akt1 and JIP1 leads to the inhibition of Akt1 activity. Most interesting, the overexpression of JIP1 led to apoptosis, as shown by cell surface blebbing and the formation of apoptotic bodies (Fig. 1 E). These observations were consistent with poly (ADP-ribose) polymerase cleavage (Fig. 1 F), which is the hallmark feature of apoptosis, and with TUNEL assay (not depicted). Fig. 1 F also shows that procaspase-9, the precursor form of caspase-9, was cleaved and activated in JIP1-overexpressing cells. Apoptosis was dependent on the level of JIP1 expression.


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 JIP1 in Akt activity in DU-145 cells. (A) Cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed, and lysates were immunoprecipitated (IP) with anti–mouse Akt1 antibody. Immunoprecipitates were analyzed for the interaction of JIP1 with Akt1 (with anti-JIP1 antibody) and Akt1 catalytic activity in vitro using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti-JIP1, anti-phospho–Ser-473–Akt1, anti-Akt1, or antiactin antibody. (B) Immunoblot of JIP1 expression in control vector transfected (pSilencer) or pSilencer-siJIP1 stably transfected (siJIP1#1–3) single cell clones from DU-145 cells. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and were immunoblotted with anti-JIP1 antibody. (C) Control plasmid or pSilencer-siJIP1 stably transfected siJIP1#2 cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt1 antibody. Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti–phospho–Ser-473–Akt1, anti-JIP1, or antiactin antibody. (D) Cells were infected with adenoviral vector containing Flag-tagged JIP1 cDNA (Ad.Flag-JIP1) at various multiplicity of infections (MOIs; 2–50). After 48 h of infection, cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt antibody. Immunoprecipitates were analyzed for Akt catalytic activity and JIP1 binding using immunoprecipitated Akt as described in Fig. 1 A (top). The presence of JIP1 or actin in the lysates was verified by immunoblotting (bottom). (E and F) Cells were infected with Ad.EGFP and/or Ad.Flag-JIP1 at various MOIs (10–200). After 48 h of infection, morphology was evaluated with a phase-contrast microscope (E), or cell lysates were immunoblotted with anti-PARP, anti–caspase-9, anti-JIP1, or antiactin antibody (F).
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fig1: Role of JIP1 in Akt activity in DU-145 cells. (A) Cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed, and lysates were immunoprecipitated (IP) with anti–mouse Akt1 antibody. Immunoprecipitates were analyzed for the interaction of JIP1 with Akt1 (with anti-JIP1 antibody) and Akt1 catalytic activity in vitro using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti-JIP1, anti-phospho–Ser-473–Akt1, anti-Akt1, or antiactin antibody. (B) Immunoblot of JIP1 expression in control vector transfected (pSilencer) or pSilencer-siJIP1 stably transfected (siJIP1#1–3) single cell clones from DU-145 cells. Lysates containing equal amounts of protein (20 μg) were separated by SDS-PAGE and were immunoblotted with anti-JIP1 antibody. (C) Control plasmid or pSilencer-siJIP1 stably transfected siJIP1#2 cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt1 antibody. Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (top). GST-Bad, phosphorylated GST-Bad, or Akt1 was detected with anti-Bad, anti–phospho–Ser-136–Bad, or anti–rabbit Akt1 antibody, respectively. Cell lysates (bottom) were immunoblotted with anti–phospho–Ser-473–Akt1, anti-JIP1, or antiactin antibody. (D) Cells were infected with adenoviral vector containing Flag-tagged JIP1 cDNA (Ad.Flag-JIP1) at various multiplicity of infections (MOIs; 2–50). After 48 h of infection, cells were lysed, and lysates were immunoprecipitated with anti–mouse Akt antibody. Immunoprecipitates were analyzed for Akt catalytic activity and JIP1 binding using immunoprecipitated Akt as described in Fig. 1 A (top). The presence of JIP1 or actin in the lysates was verified by immunoblotting (bottom). (E and F) Cells were infected with Ad.EGFP and/or Ad.Flag-JIP1 at various MOIs (10–200). After 48 h of infection, morphology was evaluated with a phase-contrast microscope (E), or cell lysates were immunoblotted with anti-PARP, anti–caspase-9, anti-JIP1, or antiactin antibody (F).
Mentions: A previous study has shown that Akt1 binds to JIP1, which is a JNK pathway scaffold protein (Kim et al., 2002). We hypothesized that JIP1 acts as a negative regulator of Akt1 and that JIP1–Akt1 interaction results in the inhibition of Akt1 catalytic activity. Metabolic oxidative stress may dissociate Akt1 from JIP1, thereby restoring Akt1 enzyme activity. To test the hypothesis, which was the first step in this study, we examined whether endogenous JIP1 associates with endogenous Akt1 and inhibits the enzymatic activity of Akt1 and whether metabolic oxidative stress dissociates Akt1 from JIP1, thereby restoring Akt1 enzyme activity. DU-145 cells were exposed to glucose-free medium for various times (10–120 min). Cells were lysed and immunoprecipitated with anti-Akt1 antibody followed by immunoblotting with anti-JIP1 antibody, or Akt1 enzyme activity was measured by using an immune complex kinase assay. Fig. 1 A shows that endogenous Akt1 interacted with endogenous JIP1 (lane 2). However, Akt1 dissociated from JIP1 within 2 h during glucose deprivation (Fig. 1 A, lane 6). Fig. 1 A also shows that Akt1 phosphorylated an Akt-specific substrate, Bad (lane 6). These results suggest that Akt1 interacts with JIP1 and that Akt1 catalytic activity is inhibited. The dissociation of Akt1 from JIP1 restores Akt1 enzyme activity, and the role of JIP1 in Akt1 catalytic activity was further examined by the knockdown of JIP1 expression. Unlike pSilencer control plasmid–transfected cells, pSilencer–short interference (si)JIP1 stably transfected siJIP1#2 cells contained a low level of JIP1 (Fig. 1 B) and promoted Akt1 enzyme activity (Fig. 1 C). In contrast, when JIP1 was overexpressed, more JIP1 associated with Akt1, and the phosphorylation of Bad on Ser-136 was inhibited (Fig. 1 D, lanes 4–6). The inhibition of Bad phosphorylation was dependent on the level of JIP1 expression (Fig. 1 D). These results suggest that the interaction between Akt1 and JIP1 leads to the inhibition of Akt1 activity. Most interesting, the overexpression of JIP1 led to apoptosis, as shown by cell surface blebbing and the formation of apoptotic bodies (Fig. 1 E). These observations were consistent with poly (ADP-ribose) polymerase cleavage (Fig. 1 F), which is the hallmark feature of apoptosis, and with TUNEL assay (not depicted). Fig. 1 F also shows that procaspase-9, the precursor form of caspase-9, was cleaved and activated in JIP1-overexpressing cells. Apoptosis was dependent on the level of JIP1 expression.

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