Limits...
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.

Show MeSH

Related in: MedlinePlus

Role of SEK1 in the interaction between JIP1 and JNK2/Akt1 during glucose deprivation. Control plasmid (pSilencer) or pSilencer-siSEK1 stably transfected (siSEK1#2) cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged JNK2 (Ad.HA-JNK2) or Ad.HA-Akt1 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A, B, D, and E) or for 60 min (C) and were lysed. Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (A–E). Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (D and E). GST-Bad or phosphorylated GST-Bad was detected with anti-Bad or anti–phospho–Ser-136–Bad antibody, respectively (top). Flag-JIP1, phosphorylated JNK, JNK2, or actin in the lysates was verified by immunoblotting with anti-Flag, anti–ACTIVE JNK, anti-JNK2, or antiactin antibody, respectively (bottom).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171369&req=5

fig7: Role of SEK1 in the interaction between JIP1 and JNK2/Akt1 during glucose deprivation. Control plasmid (pSilencer) or pSilencer-siSEK1 stably transfected (siSEK1#2) cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged JNK2 (Ad.HA-JNK2) or Ad.HA-Akt1 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A, B, D, and E) or for 60 min (C) and were lysed. Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (A–E). Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (D and E). GST-Bad or phosphorylated GST-Bad was detected with anti-Bad or anti–phospho–Ser-136–Bad antibody, respectively (top). Flag-JIP1, phosphorylated JNK, JNK2, or actin in the lysates was verified by immunoblotting with anti-Flag, anti–ACTIVE JNK, anti-JNK2, or antiactin antibody, respectively (bottom).

Mentions: We previously observed that glucose deprivation activates the ASK1–SEK1–JNK pathway (Song et al., 2002). To examine whether glucose deprivation–activated SEK1 plays a role in the interaction between JIP1 and JNK and in the dissociation of Akt1 from JIP1, we attempted to silence SEK1 expression by using short hairpin RNAs. DU-145 cells were stably transfected with either pSilencer control plasmid or pSilencer-siSEK1 vector. We selected several stable transfectants, as described in Materials and methods. We chose three transfectant clones (pSilencer, siSEK1#1, and siSEK1#2) for further studies (Fig. 6). Fig. 6 A shows that the expression of SEK1 was effectively reduced in the siSEK1#2 transfectant. To examine the role of SEK1 in the activation of JNK, pSilencer and siSEK1#2 transfectants were exposed to glucose-free medium for various times (Fig. 6 B) or for 60 min (Fig. 6 C). Fig. 6 (B and C) shows that the glucose deprivation–induced activation of JNK and the phosphorylation of p46 and p54 was suppressed in the siSEK1#2 transfectant. Relatively less suppression was observed in the siSEK1#1 transfectant (unpublished data). We further examined whether the knockdown of SEK1 mRNA and protein suppresses the interaction between JIP1 and JNK2 during glucose deprivation. Fig. 7 A shows that the binding of JNK2 to JIP1 increased during glucose deprivation in the pSilencer transfectant. However, this binding was markedly inhibited in the siSEK1#2 transfectant (Fig. 7, B and C). We also observed that glucose deprivation–induced JNK activation, the dissociation of Akt1 from JIP1, and the restoration of Akt1 activity were delayed in siSEK1#2 cells in comparison with pSilencer control vector–transfected cells (Fig. 7, E and D). These data suggest that SEK1 plays an important role in JNK2 activation, the binding of JNK2 to JIP1, and the subsequent 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)

Role of SEK1 in the interaction between JIP1 and JNK2/Akt1 during glucose deprivation. Control plasmid (pSilencer) or pSilencer-siSEK1 stably transfected (siSEK1#2) cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged JNK2 (Ad.HA-JNK2) or Ad.HA-Akt1 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A, B, D, and E) or for 60 min (C) and were lysed. Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (A–E). Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (D and E). GST-Bad or phosphorylated GST-Bad was detected with anti-Bad or anti–phospho–Ser-136–Bad antibody, respectively (top). Flag-JIP1, phosphorylated JNK, JNK2, or actin in the lysates was verified by immunoblotting with anti-Flag, anti–ACTIVE JNK, anti-JNK2, or antiactin antibody, respectively (bottom).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171369&req=5

fig7: Role of SEK1 in the interaction between JIP1 and JNK2/Akt1 during glucose deprivation. Control plasmid (pSilencer) or pSilencer-siSEK1 stably transfected (siSEK1#2) cells were coinfected with Ad.Flag-JIP1 and adenoviral vector containing HA-tagged JNK2 (Ad.HA-JNK2) or Ad.HA-Akt1 at an MOI of 10. After 48 h of infection, cells were exposed to glucose-free medium for various times (A, B, D, and E) or for 60 min (C) and were lysed. Lysates were immunoprecipitated with anti-HA antibody and were immunoblotted with anti-Flag or anti-HA antibody (A–E). Akt1 catalytic activity in vitro was determined by using GST-Bad protein as a substrate (D and E). GST-Bad or phosphorylated GST-Bad was detected with anti-Bad or anti–phospho–Ser-136–Bad antibody, respectively (top). Flag-JIP1, phosphorylated JNK, JNK2, or actin in the lysates was verified by immunoblotting with anti-Flag, anti–ACTIVE JNK, anti-JNK2, or antiactin antibody, respectively (bottom).
Mentions: We previously observed that glucose deprivation activates the ASK1–SEK1–JNK pathway (Song et al., 2002). To examine whether glucose deprivation–activated SEK1 plays a role in the interaction between JIP1 and JNK and in the dissociation of Akt1 from JIP1, we attempted to silence SEK1 expression by using short hairpin RNAs. DU-145 cells were stably transfected with either pSilencer control plasmid or pSilencer-siSEK1 vector. We selected several stable transfectants, as described in Materials and methods. We chose three transfectant clones (pSilencer, siSEK1#1, and siSEK1#2) for further studies (Fig. 6). Fig. 6 A shows that the expression of SEK1 was effectively reduced in the siSEK1#2 transfectant. To examine the role of SEK1 in the activation of JNK, pSilencer and siSEK1#2 transfectants were exposed to glucose-free medium for various times (Fig. 6 B) or for 60 min (Fig. 6 C). Fig. 6 (B and C) shows that the glucose deprivation–induced activation of JNK and the phosphorylation of p46 and p54 was suppressed in the siSEK1#2 transfectant. Relatively less suppression was observed in the siSEK1#1 transfectant (unpublished data). We further examined whether the knockdown of SEK1 mRNA and protein suppresses the interaction between JIP1 and JNK2 during glucose deprivation. Fig. 7 A shows that the binding of JNK2 to JIP1 increased during glucose deprivation in the pSilencer transfectant. However, this binding was markedly inhibited in the siSEK1#2 transfectant (Fig. 7, B and C). We also observed that glucose deprivation–induced JNK activation, the dissociation of Akt1 from JIP1, and the restoration of Akt1 activity were delayed in siSEK1#2 cells in comparison with pSilencer control vector–transfected cells (Fig. 7, E and D). These data suggest that SEK1 plays an important role in JNK2 activation, the binding of JNK2 to JIP1, and the subsequent 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