Reciprocal Negative Regulation of PDK1 and ASK1 Signaling by Direct Interaction and Phosphorylation

Hidenori Ichijo,Haiyoung Jung,Hyun-A Seong,Hyunjung Ha

doi:10.1074/jbc.m109.064295

Abstract

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H(2)O(2), tumor necrosis factor alpha, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser(967), a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H(2)O(2)-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser(394) and Ser(398), indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.

Highlights

Evidence is accumulating that the cellular decision between either cell survival or death is determined by the integration of multiple survival and death signals
It was discovered that tumor necrosis factor receptor-associated factor, Daxx, JSAP1 (JNK/ stress-activated protein kinase-associated protein 1)/JIP3 (JNK-interacting protein 3), and MPK38 stimulated ASK1 function through direct interaction, whereas thioredoxin, glutaredoxin, 14-3-3, Hsp72, Raf-1, Akt/protein kinase B (PKB), and PP5 all contributed to the inhibition of ASK1 function
PDK1 and ASK1 Interact Directly with Each Other in Vivo and in Vitro—Given that Akt, a downstream target of PDK1, phosphorylates Ser83 of ASK1 and inhibits signaling downstream of ASK1 through direct interaction and that ASK1 has been previously identified as a PDK1-interacting protein in the yeast two-hybrid system [7], we speculated that PDK1, an upstream kinase of Akt/PKB, might regulate the function of ASK1 through phosphorylation

Summary

Introduction

Evidence is accumulating that the cellular decision between either cell survival or death is determined by the integration of multiple survival and death signals. Known as protein kinase B (PKB), plays a key role in cell survival signaling and. A number of cellular proteins that interact with ASK1 have been identified, and their roles in the regulation of ASK1 function have been investigated [11,12,13,14,15,16]. Recent studies have provided evidence that the tyrosine phosphorylation of PDK1 plays an important role in stimulating PDK1 activity [25,26,27] These observations suggest that PDK1 phosphorylation is involved in the regulation of PDK1 activity, the inhibition of PDK1 function, mediated by phosphorylation, has not yet been reported. We show that PDK1 directly phosphorylates ASK1 on Ser967 through physical interaction and inactivates ASK1 This interaction contributes to the suppression of JNKmediated AP-1 (activator protein 1) transactivation and ASK1induced apoptosis. ASK1 suppresses PDK1 functions in a kinase-dependent manner by phosphorylating PDK1 at Ser394 and Ser398, suggesting a novel mechanism for the regulation of PDK1 activity

Methods

Results

Conclusion