The function of the ubiquitously expressed small GTPases RalA and RalB, which are more than 90% identical, has just recently begun to emerge. Activation of Ral can contribute to the specific biological effects induced by active Ras, such as proliferation and differentiation, depending on the cell type. For example, we recently found that Ral signaling was essential for differentiation of F9 embryonic carcinoma cells induced by Ras but not for differentiation induced by c-Jun (39
). This is epistatic evidence that Ral may have a function in a pathway that leads to c-Jun activation. Additionally, Ral and c-Jun are both targets for oncogenic Ras in cellular transformation. Therefore, we investigated whether Ras-induced c-Jun activation is mediated by Ral in A14 fibroblasts. In these cells, Ral activation is completely dependent on the activation of Ras (43
). We observed that insulin-induced phosphorylation of both serine 63 and serine 73 of c-Jun is also dependent on Ras activation. We show that an active RalGEF, but not a catalytically inactive RalGEF, induced c-Jun NH2
-terminal phosphorylation similarly to the induction by insulin treatment. Importantly, c-Jun NH2
-terminal phosphorylation in response to insulin was completely dependent on Ral activation. This pathway involves activation and phosphorylation of JNK-1 and the activation of a JNKK.
The JNKK activated by RalGTP may be JNKK-1/SEK1/MKK4, which can function as a target of activated Rac and Cdc42 in various cell lines (8
). However, in A14 cells we could not detect a clear Ral-dependent effect on JNKK-1 threonine 223 phosphorylation that is typical for this pathway. Since we did observe an increase in JNK phosphorylation in response to insulin, this may suggest that a protein kinase other than JNKK-1 functions as a target of Ral signaling. Alternatively, Ral may regulate JNKK-1 activity independent of phosphorylation. The latter model would be consistent with the observation that nonmuscle filamin (also known as ABP-280) is both a Ral GTP effector molecule and a JNKK-1 binding module (25
) and therefore may serve as a Ral-dependent scaffold protein for JNKK-1.
A number of proteins that can form a complex with Ral have been described which could be involved in the effects described in this paper. For instance, RalBP (also known as RIP or RLIP) contains GTPase activity for Rac and Cdc42 in vitro (12
). As such, RalBP may be involved in inhibiting Cdc42 signaling in a Ral-dependent manner. Perhaps RalBP allows a negative feedback between Ral and Cdc42-induced signaling toward c-Jun phosphorylation and may be important for the downregulation of JNK activity by Ral that is necessary for proper differentiation in Drosophila
). PLD1, another protein that forms a complex with Ral, plays an important role in EGF-induced cellular transformation but does not regulate JNK activity (24
). Recently, it was shown that the tyrosine kinase c-Src functions downstream from Ral (15
). Indeed, we observed that the Src tyrosine kinase inhibitor PP1 blocks insulin-induced c-Jun phosphorylation, suggesting that Src (or a Src family member) might be involved in the signaling from Ral to c-Jun. Although we cannot exclude the possibility that the PP1-sensitive Ral target is distinct from Src or related tyrosine kinases, PP1 has been described as a highly specific inhibitor of this family of kinases (17
). The way Ral activates Src and the way Src activates JNK are currently unclear. Interestingly, a physiological link between Src and JNK also exists in Drosophila
; i.e., activation of the JNK homolog Basket (Bsk) functions downstream of Src in epidermal closure (37
JNK activation has also been implicated in signaling in response to cellular stresses and the subsequent onset of apoptosis (1
). However, Ral is not activated in response to cellular stresses (data not shown). In addition, we did not find any effect of dominant negative Cdc42-N17 on insulin-mediated c-Jun phosphorylation, excluding a prominent role for Cdc42 in the insulin-Ral pathway leading to phosphorylation of c-Jun. It is clear that the effects of c-Jun on cellular responses will depend strongly on the cell type and cellular environment (23
). Importantly, c-Jun is required for Ras-induced transformation and proliferation of rodent fibroblasts and plays a determinant role in the regulation of normal mammalian development (23
). Our results can provide an explanation for the overlapping effects of Ral signaling and activation of c-Jun, indicating that regulation of c-Jun can at least in part explain the biological effects of Ral signaling. c-Jun is not the only transcription factor that is under control of the Ras-Ral pathway. For example, the fork head transcription factor AFX is regulated by phosphorylation in response to Ral signaling and insulin treatment (22
). Phosphorylation of AFX plays an important role in Ras-dependent cell cycle control and transformation (26
In conclusion, in this paper we describe a signal transduction pathway that couples growth factor signaling to the phosphorylation of c-Jun. This pathway involves Ral, presumably Src, and a still elusive JNKK. Ral-mediated phosphorylation of c-Jun and AFX, transcription factors that critically determine cellular growth responses, firmly establishes that the Ral pathway plays an important role in transcriptional regulation downstream of Ras.