In PC12 cells, constitutive Ras activity promotes cell cycle arrest and differentiation into a neuronal phenotype. This phenotype is mediated in part by Ras-induced Raf activation, since constitutively activated versions of Raf can mimic, at least to some extent, the effects of activated Ras (53
). Moreover, inhibition of the Ras-Raf-Erk pathway blocks stimulus-induced neurite outgrowth (40
). Similar conclusions have been reached for Ras-induced PI3 kinase activity (16
). Remarkably, we have shown here that a third class of downstream Ras target, Ral-specific exchange factors, promotes the opposite effect. In particular, transfection of either a mutant Ras12V37G that preferentially activates Ral-GEFs over Raf or PI3 kinase, or an isolated catalytic domain of a Ral-GEF (Rgr), suppressed neurite outgrowth induced by NGF. Importantly, not all NGF signaling was blocked, since NGF treatment still enhanced fos
promoter activation in Rgr-expressing cells. Rgr expression also suppressed neurite outgrowth promoted by a Ras mutant (12V35S) that preferentially activates Raf, documenting that Ral-GEF signaling antagonizes the action of other Ras effector pathways in this system.
Importantly, transfection of a dominant negative Ral28N mutant, which is thought to prevent the catalytic domain of all Ral-GEF family members from activating endogenous Ral proteins, enhanced NGF induction of neurite outgrowth. Again, this is the opposite of what is observed when Ras-induced Raf or PI3 kinase activities are blocked in PC12 cells. Moreover, not all signaling via NGF receptors was altered, because NGF induction of fos gene activation was affected little if at all in cells expressing Ral28N. Together, these findings show that one potential function of Ral-GEF signaling in PC12 cells is to suppress neurite outgrowth.
Since NGF induction of neurite outgrowth is associated with cell cycle arrest, a possible mechanism involved in Ral-GEF action could be the prevention of cell cycle exit. Alternatively, Ral-GEF activity could allow cell cycle arrest but inhibit cellular machinery involved in neurite outgrowth, such as the actin cytoskeleton. The latter mechanism is plausible because a putative Ral target protein, RalBP1, is a GAP for the actin-regulating CDC42 and Rac GTPases. Support for the first model came from the observation that changes in Ral-GEF activity dramatically affected the cell cycle in a manner consistent with observed effects on neurite outgrowth. For example, expression of Ras37G or the Ral-GEF Rgr suppressed cell cycle exit upon NGF stimulation. In some experiments, NGF stimulation was performed during serum starvation, so that Ral-GEF activity overcame two growth-suppressing signals. When Rgr was expressed in differentiated cells, existing neurites remained intact, suggesting that the primary effect of the Ral pathway in this system is to influence the formation of neurites rather than their maintenance. However, it is likely that Ral-GEF proteins play an additional role in fully differentiated neurons, since Ral GTPases are expressed at high levels at nerve endings (2
Inhibition of endogenous Ral-GEFs by expression of dominant negative Ral28N had the opposite effect. It promoted cell cycle exit in growing populations of PC12 cells as efficiently as NGF treatment did. However, unlike NGF treatment, expression of Ral28N was not sufficient to promote neurite outgrowth. Ral28N expression also accelerated cell cycle exit induced by NGF treatment of cells growing in serum, which is consistent with its ability to enhance NGF-induced neurite outgrowth under these conditions. Serum starvation also accelerated cell cycle exit. Like Ral28N, serum starvation also was not sufficient to promote neurite outgrowth. This suggested that Ral28N expression and serum starvation might both work solely by enhancing cell cycle exit. However, Ral28N expression in serum-starved cells had little additive effect on cell cycle exit, yet it still dramatically enhanced neurite outgrowth induced by NGF (Fig. ). Apparently, inhibition of Ral-GEF promotes neurite outgrowth by an additional mechanism.
To enhance Ral-GEF activity in cells, we transfected either a Ras mutant shown to preferentially activate Ral-GEFs or an isolated catalytic domain of the Ral-GEF Rgr (4
). The only known function of this fragment of Rgr is Ral activation, so it is likely that some function of this GTPase is responsible for suppressing cell cycle arrest induced by NGF. However, we did not observe comparable inhibition (or enhancement) of NGF-induced neurite outgrowth by expression of constitutively activated Ral mutants (data not shown). The fact that a Ral-GEF displays stronger biological activity than a GTPase-deficient version of its target protein has also been observed in fibroblasts (36
). In that system, Ral-GEF activity complements fos
gene activation and growth-promoting functions of Raf more efficiently than activated Ral. The reasons for this unusual phenotype are not yet known. Thus, we cannot completely exclude the possibility that the catalytic domain of the Ral-GEF used here, and Ral-GDS, RGL, and Rlf used in the experiments on fibroblasts, actually activate a GTPase different from Ral.
Ral GTPases are capable of interacting with at least two classes of signal transduction molecules that could potentially account for the effects on PC12 cells observed here. One is a PLD, and the other is a GAP for Rac and CDC42 GTPase. Ral proteins can bind to PLD1 and augment its activation by the Arf GTPase (18
). Another protein capable of activating PLD1 is the Rho GTPase (1
). Interestingly, like that of Ral-GEFs, Rho activity also inhibits neurite outgrowth, and suppression of Rho activity enhances neurite outgrowth. Thus, we investigated whether cooperative regulation of PLD1 by Rho and Ral might account for the similar phenotypes of the two GTPase pathways. However, our data do not support this model. First, transfection of a mutant Ral lacking the segment required for PLD binding did not enhance neurite outgrowth. This mutant has been shown to block tyrosine kinase activation of PLD in fibroblasts and would be expected to mimic the effects of Ral28N expression if PLD activation was involved (18
). Second, neurites induced by expression of Ral28N were still sensitive to neurite retraction induced by LPA, which functions through Rho activation. Ral28N-induced neurites would be resistant to LPA if active Ral and Rho were both required to inhibit neurite outgrowth through activation of a common PLD. Finally, activation of Rho causes retraction of fully formed neurites, whereas expression of Rgr did not.
We did find evidence supporting a role for CDC42 and Rac in Ral-GEF action. These GTPases have been implicated in Ral function by the observation that the Ral target, RalBP1, is a CDC42 and Rac GAP and thus has the potential to negatively regulate these proteins. A simple hypothesis is that Ral-GEFs activate Ral and then Ral activates the GAP activity of RalBP1, possibly by targeting it to Ral-containing membranes, where a fraction of active CDC42 and Rac might exist (Fig. ). This could explain, at least in part, the neurite-inhibitory effects of Ral-GEF overexpression, since suppression of CDC42 or Rac activity (by expression of dominant inhibitory mutants of these GTPases) has been shown previously (and here) to inhibit neurite outgrowth (26
). In addition, we found that expression of these dominant negative mutants mimicked the action of Rgr by suppressing NGF-induced cell cycle exit. We also found that expression of constitutively activated CDC42 or Rac mutants, which are insensitive to GAP proteins, at least partially reversed Rgr action.
FIG. 7 Ras effector pathways in NGF stimulated PC12 cells. Constitutive activation of Raf or PI3 kinase promotes cell cycle arrest and differentiation of PC12 cells into a neuronal phenotype. In contrast, constitutive activation of Ral-GEF promotes proliferation (more ...)
Similarly, inhibition of Ral-GEF activity by Ral28N expression would be expected to potentiate CDC42 and Rac activity by suppressing RalBP1 activity (Fig. ). This might explain some of the neurite-enhancing effects of Ral28N expression, since constitutively activated CDC42 and Rac have been shown here and previously by others to potentiate NGF-induced neurite outgrowth (26
). Our observation that dominant negative CDC42 or Rac suppressed the effects of Ral28N on neurite outgrowth is also consistent with this model. Since RalBP1 has the capacity to bind to two highly related Eps homology domain proteins, Reps1 and POB (15
), these proteins may also be involved in regulating neurite outgrowth in PC12 cells.
We have shown that Ras12V37G and Rgr expression can transactivate a reporter construct containing the regulatory sequences of the c-fos
gene. A similar result was observed previously in fibroblasts by transfection of Ras37G (50
) or the Ral-GEFs Ral-GDS, RGL, and Rlf (33
). Therefore, it is likely that Ral-GEF activity regulates PC12 cell function, at least in part, by altering gene expression patterns. However, discovery of the activation of the fos
gene promoter in PC12 is not particularly revealing, since expression of Ras12V35S, which produces an effect which is the opposite of that of Rgr, also activates the fos
promoter under the same conditions. Presumably, Ral-GEFs and Raf activate the fos
promoter by different mechanisms and have the capacity to activate or inhibit different subsets of genes. Identification of Ral-GEF-specific alterations in gene expression may help reveal how these two signaling pathways induce opposing effects in PC12 cells.
Much attention has been focused on the Ras-induced Raf-Erk kinase cascade as a dominant growth-promoting branch of the Ras effector signaling system. However, the results reported here in PC12 cells, along with previous results in fibroblasts and thyrocytes, highlight novel features of the growth-promoting activity of the Ras-induced Ral-GEF signaling cascade. First, the Ral-GEF we used in these studies, Rgr, was isolated as an oncogene whose protein product is capable of causing 3T3 cells to form tumors in animals, despite their lack of focus-forming activity in tissue culture (4
). Second, expression of a constitutively activated Rlf led to enhanced growth rates of 3T3 cells in culture (50
). Third, thyroid-stimulating hormone promoted cell proliferation in thyrocytes through a Ras- but not Raf-dependent signaling pathway. Expression of Ras37G mimicked this effect, and Ral28N expression blocked it, suggesting that Ras-induced activation of Ral-GEFs was responsible (32
). Finally, we show here that constitutive Ral-GEF activity promotes cell proliferation in NGF-treated PC12 cells while constitutive Raf and PI3 kinase activities promote cell cycle arrest and differentiation. Thus, elevated Ral-GEF activity can clearly promote cell proliferation. In fact, in the last two cell systems described above, cell proliferation is more tightly coupled to Ral-GEF activation than to Raf activation.
Recent studies document that Ral proteins can be activated by both Ras-dependent and Ras-independent pathways. Ras-dependent pathways are thought to be mediated by a family of Ral-GEFs that can bind to and be activated by Ras-GTP (11
). The mechanism underlying Ras-independent Ral activation is not well understood beyond the fact that it can be initiated by elevated levels of calcium (14
). We have shown here that NGF can lead to elevated levels of RalB and, to a lesser extent, RalA. We attempted to determine the Ras dependence of this event by using a PC12 cell line whose endogenous Ras exchange factors are blocked by expression of the dominant negative Ras17N mutant. However, we detected residual Ras activation upon NGF stimulation, which made firm conclusions difficult. Nevertheless, the data suggest that Ras-dependent and Ras-independent mechanisms exist. Ras dependence was suggested by the fact that the extent and duration of Ral activation was reduced in the mutant cell line. Ras-independent Ral activation was suggested by the fact that a large decrease in Ras activation led to only a small decrease in Ral activation. Clearly, additional studies will have to be employed to dissect what may be a complicated mechanism of Ral regulation.
An important conclusion from this study is that the ratio of Ral-GEF, Raf, and PI3 kinase activities can determine whether PC12 cells proliferate or differentiate, with Ral-GEF promoting the former and Raf and PI3 kinase promoting the latter. The Raf-Erk signaling pathway can also promote cell cycle arrest in other cell types, such as primary fibroblasts (42
), suggesting that this concept may be valid in other cell systems. The results reported here also show that the ratio of signals emanating from Raf and Ral-GEFs changes during PC12 cell stimulation. In particular, NGF activates both Erk and RalB acutely. However, Erk activation persists for hours, while Ral-GTP levels subside after ~20 min. Thus, Ral activation likely contributes primarily to the early effects of NGF on PC12 cells, where it serves to delay the onset of cell cycle arrest and differentiation induced by NGF. The rapid inactivation of the Ral signaling pathway appears to be necessary to permit subsequent NGF-induced cell cycle arrest and differentiation. Consistent with this notion is our observation that EGF, which can enhance the proliferation of PC12 cells, also promotes Ral activation transiently.
Why Ral is unresponsive to Ras after ~20 min of NGF stimulation remains to be determined. It is intriguing that recent results suggest that Raf also becomes uncoupled from Ras after acute activation in these cells (56
). Sustained activation of Erk in response to NGF was reported to be due to NGF activation of the Ras-related GTPase Rap1, which in turn was shown to activate B-Raf. Interestingly, existing evidence indicates that Rap does not activate Ral-GEFs in most cell types (46
). It is tempting to speculate that Rap replaces Ras in activating Erk in the late stage of NGF signaling in PC12 cells, at least in part, to avoid sustained Ral-GEF activation.