Akt plays a role in cell survival through phosphorylation of Bad on Ser136
). It is likely that Akt promotes cell survival through mechanisms other than phosphorylation of Bad (27
). For example, Akt also phosphorylates and inactivates caspase 9 and members of the family of Forkhead transcription factors (8
). We previously demonstrated that c-N-Ras promotes cell survival through an Akt-dependent pathway that results in the phosphorylation of Bad (129
). Furthermore, we find that this c-N-Ras-dependent survival function is operational at steady state in the absence of any treatments. It was presumed that this c-N-Ras/Akt survival pathway functions through the direct interaction of c-N-Ras with PI3-kinase, the upstream activator of Akt. In fact, PI3-kinase interacts with Ras in vitro in a GTP-dependent manner (29
). More recently, however, a stable Ras-PI3-kinase complex has been observed in FRTL5 cell lysates only after treatment of the cells with cyclic AMP-elevating agents (15
). We believe that the survival function of c-N-Ras in producing steady-state phosphorylation of Akt and Bad proceeds through one of the PI3-kinase isoforms, since these phosphorylation events can be downregulated through the use of PI3-kinase selective inhibitors (Fig. ). It is unclear whether this c-N-Ras-dependent survival function proceeds through a direct interaction between c-N-Ras-GTP and PI3-kinase.
Our data suggest that c-N-Ras promotes cell survival through both Akt-dependent and Akt-independent mechanisms. The possibility that JNK may play a role in apoptosis prompted us to examine whether c-N-Ras provides a survival function through the regulation of JNK activation. The biological outcome of JNK signaling and its role in apoptosis remain controversial and may depend upon the nature of the environmental stimulus as well as the cell type and the presence of other regulatory factors (48
). Emerging evidence indicates that whether the activation of JNK is pro-survival or pro-apoptosic may depend on the magnitude and duration of JNK activation. There are several reports which provide evidence indicating that persistent, rather than transient, JNK activation or activity is associated with apoptosis (13
). Our data are consistent with the concept that elevated and sustained phosphorylation of JNK is proapoptotic. Cells that are more resistant to the induction of apoptosis (control N+/+
and c-N-Ras reconstituted cells) display only transient JNK phosphorylation and activity. Cells that are more apoptotically sensitive (N-Ras knockout cells) demonstrate prolonged activation of JNK. In fact, artificially elevating and prolonging the duration of JNK activation resulted in increased apoptotic sensitivity [N+/+
(1)/MEKK3:ER] (Fig. ). Our data therefore suggest that c-N-Ras provides protection from apoptosis through the attenuation of the magnitude and duration of JNK activity (Fig. ).
The activation of p38 plays a role in the apoptotic process in some cell types (9
). As has been observed with JNK, in some cases elevated and sustained activation of p38 correlates with apoptosis (33
). We found similar results when we examined p38 activation. Our data suggest that, as observed with JNK, elevated and prolonged activation of p38 is associated with apoptosis and that c-N-Ras functions to promote cell survival through attenuation of the magnitude and duration of p38 activation (Fig. ).
One of the major questions in the present study is the nature of the mechanism by which c-N-Ras regulates JNK and p38 activation. Three possibilities in answering this question exist: (i) c-N-Ras reduces the activation of upstream kinase activators of JNK and p38; (ii) c-N-Ras upregulates a JNK- or p38-specific phosphatase; or (iii) a combination of the two mechanisms. There are a limited number of upstream dual-specificity kinase activators of JNK and p38; they include MKK4 and MKK7 for JNK activation (21
), and MKK3 and MKK6 for p38 activation (24
). MKK4 is also capable of activating p38 as well as JNK (24
). All four kinases activate JNK or p38, respectively, through phosphorylation of both a threonine and a tyrosine residue.
We investigated the activation kinetics of these upstream JNK/p38 activators by examining their phosphorylation state both at steady state and following treatment with apoptotic agonists. The data reveal a pattern of MKK protein phosphorylation that parallels the results obtained with JNK and p38 activation. Cells that are apoptotically sensitive (N-Ras knockout) display elevated and sustained activation of upstream kinase activators for both the JNK and p38 signaling pathways. Cells that are resistant to the induction of apoptosis (control N+/+ cells and c-N-Ras reconstituted cells) possess only transient activation of either MKK4 or MKK3/6. These results were confirmed by indirect measurement of the activities of the upstream kinase activators by using recombinant JNK and p38 in the in vitro double-kinase assays (Fig. ). Taken together, these results indicate that c-N-Ras promotes cell survival by downregulating JNK and p38 activation through a mechanism involving c-N-Ras-dependent downregulation of the upstream dual-specificity kinase activators of both JNK and p38. These results also suggest that c-N-Ras functions at a position that is a branch point upstream of all of the MKK proteins, whereby c-N-Ras prevents sustained activation of the MKK proteins that function in both the JNK and p38 signaling pathways.
While the JNK group of MAP kinases are strongly stimulated by extracellular stress and inflammatory cytokines, they can be modestly activated by growth factors (36
). We treated cells with EGF to investigate whether the N-Ras knockout cells exhibit elevated JNK activity because they were unable to downregulate JNK. We observed transient phosphorylation of JNK in all of the cell lines (Fig. ). These data suggest that transient activation of JNK is not associated with apoptosis and that the N-Ras knockout cells are capable of downregulating JNK activity. We have not ruled out the possibility that different inactivation mechanisms might be involved in JNK downregulation, one of which might be deficient in the N-Ras knockout cells. However, our data clearly demonstrate a failure to inactivate JNK/p38, as well as their upstream activators, following apoptosis induction in the absence of c-N-Ras.
It therefore appears that c-N-Ras promotes cell survival through upregulation of Akt activity and downregulation of JNK. Our data also suggest that c-N-Ras functions at a branch point upstream of the MKK activators of both the JNK and p38 signaling pathways. We therefore chose to go downstream of N-Ras to determine the effector(s) through which N-Ras causes downregulation of JNK activation. We stably expressed three different switch 1 effector domain mutant N-Ras constructs in the N-Ras knockout cells. We were surprised to find that only the RalGDS-binding N-Ras mutant (37G) was capable of both restoring JNK regulation and rescuing the apoptotically sensitive phenotype of the parental N-Ras knockout cells. While these results were unexpected, experiments using the MEK-specific inhibitor U0126 have indicated that inhibition of the Erk/MAP kinase pathway does not lead to a further increase in the apoptotic sensitivity of either the N-Ras knockout or control cells (data not shown). We have also demonstrated that inhibition of PI3-kinase in the control N+/+ cells does not, by itself, increase their apoptotic sensitivity (Fig. ). These data suggest that at least one cellular survival mechanism is the downregulation of JNK signaling, which proceeds through a mechanism that is independent of either Raf-1 or PI3-kinase (p110α or p110β). We have also found that the 37G switch 1 mutant N-Ras restores JNK regulation in the presence of PI3-kinase inhibitors (data not shown). This, then, suggests that attenuation of JNK activity by N-Ras occurs through a unique set of downstream effectors independent of Raf-1 and PI3-kinase.
One concern was the failure of the PI3-kinase-binding N-Ras mutant to rescue the apoptotic sensitivity of the N-Ras knockout cells. A large body of evidence supports a survival role for PI3-kinase and Akt (4
). While one of the survival functions of c-N-Ras is the restoration of Akt activity, it is unclear whether this function is the result of a direct interaction between c-N-Ras and PI3-kinase. Further, we have evidence suggesting that a direct interaction between oncogenic Ha-Ras and PI3-kinase is dispensable for G12V-Ha-Ras-dependent transformation of IEC-6 epithelial cells but not for their migration toward soluble fibronectin (49a
). It is conceivable that in some cases Ras may lead to the activation of PI3-kinase and Akt by indirect mechanisms. Another possibility is that c-N-Ras provides its steady-state antiapoptotic function through the p110δ subunit of PI3-kinase. While the p110α isoform reportedly binds only to the 40C switch 1 Ras mutant, p110δ binds to the 37G and not the 40C mutant Ras (60
). This, then, suggests the possibility that c-N-Ras might interact with only the p110δ catalytic subunit of PI3-kinase to provide an antiapoptotic signal. Nevertheless, our data strongly support a survival function of c-N-Ras through a direct interaction with RalGDS, AF-6, Rin 1, PLC
, or p110δ, which leads to the downregulation of JNK activation and cell survival.
It is of interest that the Raf-1-binding mutant 35S does not rescue the apoptotically sensitive phenotype of the parental N-Ras knockout cells at levels of expression that are significantly above that observed for the c-N-Ras in the control N+/+
cells (Fig. ). The PI3-kinase-binding mutant 40C does not reverse the parental N-Ras knockout cell phenotype at levels that are either below or above the level of c-N-Ras in the control N+/+
cells. In contrast, the 37G, “RalGDS/AF-6/Rin 1/PLC
/p110δ”-binding N-Ras mutant rescues the apoptotic sensitivity of the parental N-Ras knockout cells at levels of expression that are considerably below the level of c-N-Ras present in the control cells (Fig. , 37G clones 4 and 5). Expression of c-N-Ras in the N-Ras knockout cells also rescues the apoptotic sensitivity of the parental N-Ras knockout cells at levels of expression that are nearly identical to the levels of c-N-Ras in the control N+/+
). These results suggest that if a protein expressed in cells is going to perform a particular function, it is likely that it will do so without the need for vast overexpression. In fact, some of the complexity that revolves around interpreting Ras function may result from overexpression versus expression at endogenous levels or transient expression versus stable expression (106
In summary, our results indicate that c-N-Ras promotes cell survival through multiple mechanisms. c-N-Ras provides protection from apoptosis by the upregulation of Akt activity and downregulation of the magnitude and duration of JNK and p38 activity. Recent evidence supports the concept that multiple pathways contribute to Ras-dependent cell survival (71
). While we cannot rule out the contribution of other, as yet unidentified, effectors, it appears that c-N-Ras promotes cell survival through upregulation of Akt activity and through a RalGDS/AF-6/Rin 1/PLC
/p110δ-dependent mechanism that leads to downregulation of signaling through JNK and p38.
N-Ras provides a steady-state survival signal in a nonclassical signaling environment. Our previous work and the data described in this report support the concept that Ras proteins, or at least c-N-Ras, possess functions other than to regulate acute proliferative signaling. The best illustration of tonic Ras-dependent signaling is the reaction to serum withdrawal. Removal of serum, which is usually associated with inactivation of Ras proteins, results in increased apoptosis in the N-Ras knockouts but not in control N+/+ cells. This, then, implies that c-N-Ras, in the absence of serum, is providing a functional antiapoptotic signal. This suggests the possibility of a novel Ras paradigm that is distinct from the control of acute, proliferative signal transduction.