Understanding mechanisms underlying cross-talk among signal transduction pathways is key to unveiling the dynamics of multidimensional regulatory signaling networks. Although such networks fine-tune cellular function under normal growth as well as following stress and DNA damage, it has been a challenge to understand the nature of changes in this complex mechanism that occur in pathological cases, including human cancer. Melanoma, an aggressive form of skin cancer that often harbors mutant BRAF or N-RAS, and consequently increased ERK-MAPK activity, serves as a paradigm of re-wiring signaling pathways.
Here we provide a blueprint for a re-wired signaling pathway in melanoma. Our data reveal that ERK increases the level of c-Jun expression by affecting its transcription and stability. We further demonstrate that c-Jun increases the transcription of RACK1, an adaptor protein required for activation of JNK by PKC, which constitutes a feed-forward mechanism that increases c-Jun transcriptional activity. Lastly, we demonstrate that the ERK-Jun signaling cascade is required for c-Jun-mediated transcription of cyclin D1, which is often found to be overexpressed in human melanomas. Analysis of melanoma tumors confirms the changes in these signaling pathways, for which our studies provide the underlying mechanisms.
Our study identifies the mechanisms underlying upregulation of c-Jun expression by ERK, as we demonstrate the effect of ERK on GSK3 inactivation, resulting in c-Jun protein stabilization, and on CREB activation, which increases c-Jun transcription (model proposed in ). Activation of ERK results in phosphorylation of CREB at Ser133, either directly by ERK or by ERK-stimulated MSK (Wiggin et al., 2002
). The finding that ERK contributes to stabilization of c-Jun through inactivation of GSK3 is consistent with those reported by Wei et al. (2005)
, who demonstrated that phosphorylation of c-Jun on T239 by GSK3β is critical in c-Jun degradation. Yet whereas Akt was shown to affect GSK3β phosphorylation in HeLa cells (Wei et al., 2005
), in melanoma cells, ERK signaling was found to mediate primarily GSK3 phosphorylation, resulting in c-Jun stabilization. ERK-dependent GSK3 phosphorylation at S9/S21 is likely to be mediated by an intermediate kinase, although we have ruled out RSK (siRNA of RSK did not impact c-Jun stability in these cells; data not shown). Alternatively, association of ERK with GSK3 and subsequent phosphorylation of GSK3 at T43 was shown to enhance the rate of phosphorylation on Ser9 by a third kinase (Ding et. al, 2005
Model of ERK cross-talk with JNK signaling
Our data identify RACK1 as a c-Jun transcriptional target. It is plausible that other c-Jun target genes may contribute to the feed-forward loop mechanism. Among those are PDK1, the PKC upstream kinase, whose transcription also appears to be c-Jun-dependent (Lopez-Bergami et al., unpublished data) and c-Jun-dependent changes in expression of protein phosphatases that affect JNK activity (Sprowles et al., 2005
). Several requirements must be satisfied for the feed-forward loop to exist. First is that c-Jun is also subjected to phosphorylation on residues S63, S73 (and possibly T91 and T93), thus acquiring its transcriptional capabilities. Such phosphorylation is primarily attributed to JNK, although it has been also ascribed to ERK in JNK-deficient cells (Morton et al., 2003
); our data rule out ERK involvement in this process in melanoma (Fig. S5; S6
), further substantiating the need for the ERK-Jun link with subsequent activation of the RACK1-JNK-PKC module. The second requirement is that JNK must be activated by the canonical MKK4/7 pathway. This requirement is crucial since the contribution of PKC to overall JNK activity depends on JNK’s own phosphorylation on amino acids 183/5 by MKK4/7. Since active JNK is commonly seen in melanoma (; ; Lopez Bergami et al., 2005b
), this requirement is satisfied, although the reason for the upregulation of JNK in melanoma is not completely clear. The finding that some tumor samples display positive P-JNK staining in the absence of ERK activity suggests that JNK activation proceeds through ERKindependent mechanisms. In any case, in agreement with our model, JNK activity by itself is not sufficient to maintain high levels of c-Jun. The third requirement relates to PKC’s own activity. To cooperate with RACK1 in augmenting JNK activity, PKC must be active. Earlier studies pointed out that expression of several PKC isoforms is elevated in melanoma (Oka and Kikkawa, 2005
; Selzer et al., 2002
). Here we demonstrate that PKCα/β are among the isoforms that appear to be predominantly active in melanoma ( and Fig. S17
). That PKC is important for growth and metastasis of melanoma was shown in studies where pharmacological inhibitors of PKC effectively inhibited growth of melanoma in mouse xenograft models (Dumont et al., 1992
; Mapelli et al., 1994
). That PKC and JNK are active in this tumor type satisfies the above-stated requirements and supports the existence of the feed-forward loop mechanism triggered by ERK’s effect on c-Jun expression levels ().
The cell lines we used were obtained, for the most part, from vertical growth phase tumors (A375, WM115, Lu1205, WM9), which are associated with more metastatic phenotypes. Consistent with our findings, CREB activity was implicated in melanoma growth and metastasis (reviewed in Nyormoi and Bar-Eli, 2003
). Similarly, via its heterodimerization with ATF2, c-Jun was also shown to play an important role in melanoma growth (Bhoumik et al., 2004
). Importantly, regulation of c-Jun by the ERK pathway was not seen in cells that lack constitutively high levels of ERK activity (e.g., melanocytes or HEK293 cells), suggesting that this regulation reflects selective rewiring of melanoma tumors in which upstream MAPK are constitutively active. It is likely that our finding extends beyond the cases of mutant B-RAF and N-RAS since activation of the MEK/ERK pathway and upregulation of cyclin D1 are also seen in tumors where such mutations do not exist. Thus, one would expect that the newly established link between ERK and JNK, with its implications regarding cyclin D1 expression, may allow identifying additional deregulated signaling components along the JNK or ERK signaling pathways. One would also expect that the link between ERK and JNK would also exist in other tumors where ERK is upregulated, a common occurrence in human cancer (Davies et al., 2002
; Downward, 2003
The effect of ERK on both transcriptional activation and stabilization of c-Jun via CREB activation and GSK3 inactivation, respectively points to the importance of securing high levels of c-Jun expression, as illustrated in melanoma-derived cells. The fact that two pathways cooperate in increasing c-Jun expression levels reflects independent mechanisms whose impact on c-Jun expression might involve different kinetics and possibly magnitudes (Murphy and Blenis, 2006
). This consideration may be important in fine-tuning the degree of inhibition of ERK activity in melanomas where ERK is among the primary targets of therapy.
In summary, the study presented here provides an undisclosed insight into the mechanism underlying the link between the ERK and JNK pathways that is mediated by ERK’s effect on c-Jun expression levels. This re-wiring has been demonstrated in human melanoma, where mutant N-RAS or B-RAF affect downstream ERK signaling. In providing an initial blueprint for re-wiring key signal transduction pathways in melanoma, our findings point to additional targets for therapy of this tumor type.