Our results suggest that ASK1 activity and consequent activation of downstream signaling molecules can be negatively regulated by Akt stimulation. In support of this hypothesis, Akt phosphorylated ASK1 on serine 83, a site similar to the consensus sequence previously identified for other Akt substrates. An association between Akt and ASK1 in cells endogenously expressing these two proteins further suggests a functional link between these kinases. The interaction between these proteins is reminiscent of the Akt-BAD interaction observed in transfected cells (12
). ASK1, therefore, can interact constitutively with Akt, but ASK1 phosphorylation is influenced by Akt activity. Collectively, the phosphorylation and coimmunoprecipitation results suggest that ASK1 is an Akt substrate.
Support for a physiological role for Akt-mediated ASK1 phosphorylation comes from several lines of evidence. First, Akt was able to suppress ASK1's response to oxidative stress. Akt, however, had little effect on the H2O2 responsiveness of the ASK1 point mutant, ASK1S83A, suggesting a specific phosphorylation event in Akt-mediated inhibition. Moreover, in cells endogenously expressing ASK1 and Akt, ASK1 activation by serum deprivation was inhibited by IGF-1 in a manner dependent on PI3-K/Akt activity. In confirmation of these findings, the kinase activity of highly expressed ASK1 was decreased by Akt coexpression, and this inhibition was also dependent on ASK1 serine 83. The moderate extent of ASK1 inhibition by Akt (30%) under this stimulus-independent condition perhaps reflects the decreased responsiveness of high ectopic ASK1 to physiological regulation. Indeed, in our experiments, the kinase activity of highly overexpressed ASK1 could not be stimulated further by H2O2 exposure (data not shown).
Modification of ASK1 function would be predicted to alter JNK- and p38-mediated gene transcriptional events as well as cell viability according to the cellular context. The JNK and p38 kinase cascades can initiate or modify transcription by activating several transcription factors, such as members of the AP-1 family and ATF-2 (30
). A constitutively active ASK1 has, for instance, been shown to induce differentiation through p38 activation in PC12 cells (40
). Given Akt's inhibitory effects on ASK1-mediated JNK and ATF-2 activities, Akt regulation of ASK1 may therefore represent an additional role for Akt in transcriptional control. Akt was initially suggested to influence gene transcription because of its ability to translocate into the nucleus (2
). More recently, Akt has been demonstrated to activate transcription factors, such as NF-κB and CREB (15
An alternative—but not mutually exclusive—role for Akt in regulating ASK1 function may be to suppress ASK1-induced cell killing. In our experiments, we found that ASK1 phosphorylation by the PI3-K/Akt pathway could reduce apoptosis induced by transfected ASK1 in 293 and HeLa cells. The cell death induced by ASK1 in certain contexts may, therefore, be ameliorated by simultaneous Akt activation. For instance, in superior cervical ganglion neurons, which die in an ASK1-dependent manner upon withdrawal of nerve growth factor, nerve growth factor-induced survival may in part reflect activation of Akt and subsequent inhibition of ASK1 (11
Akt suppression of ASK1 remains to be examined in the context of other ASK1 regulators. Among the negative regulators are thioredoxin and the cell cycle inhibitor, p21Cip1/WAF1
). The adapter protein, 14-3-3, interacts with ASK1 and inhibits cell killing without altering ASK1 kinase activity (50
). Certain TNF receptor-associated factor (TRAF) proteins, such as TRAFs 2, 5, and 6, and ROS can activate ASK1, at least in part, through ASK1 dimerization (18
). Both thioredoxin and TRAF2 have been shown to interact with the ASK1 N terminus, a region that contains the Akt consensus site. Akt phosphorylation of ASK1 may therefore alter the binding affinity of ASK1 for TRAF2 or thioredoxin. It is also conceivable that Akt phosphorylation of ASK1 cannot occur unless TRAF2 or thioredoxin dissociation from ASK1 occurs first. The relative impact of these and other ASK1 regulatory proteins on ASK1 function may well be cell context specific.
Other molecular targets of Akt may also be responsible for antagonizing the activity of the stress-activated kinase cascades in certain cell contexts. Rac1, a small G protein activator of the JNK pathway, has been reported to be phosphorylated by Akt, an event which leads to decreased Rac1 GTP-binding affinity (29
). However, this effect was directly tested in vitro only, and the outcome of Rac1 phosphorylation on downstream targets in intact cells has not yet been determined.
Given the growing number of Akt substrates, a central question of Akt signaling is whether multiple substrates can be simultaneously phosphorylated by Akt in a given system. To our knowledge, no integrated investigation of the Akt effectors has been carried out in one system. In this vein, we have conducted preliminary coimmunoprecipitation experiments, which demonstrated that Akt immunoprecipitated from L929 cells associates with both Raf-1 and ASK1, suggesting that these Akt substrates are positioned to undergo phosphorylation at the same time (data not shown). Therefore, Akt may have the potential to deploy several of its reported downstream functions in parallel, perhaps acting as a central regulator in a multipathway signaling complex. Clearly, a more thorough characterization of the kinetics and subcellular localization of Akt and its different substrates, as well as more genetic evidence, will be required to assess the relative contribution of a given substrate to Akt function in vivo.
Our results suggest that one point of convergence between the Akt pathway and the stress-activated kinases is ASK1. The involvement of Akt in the inhibition of apoptosis regulatory protein kinases broadens the scope of Akt as a transcriptional modifier and mediator of cell survival decisions.