A marked increase in the incidence of melanoma has been accompanied by greater interest to elucidate mechanisms underlying the development of this cancer. The previous work led to the identification of c-jun, CREB and ATF2 as transcription factors that play a key role in melanoma development. Close examination of the regulation of these transcription factors revealed that melanoma cells contain a low-MW transcriptional inhibitor that is induced by UV-irradiation. He has successfully identified the nature of this transcriptional inhibitor as ubiquitin (Ub). Ubiquitination of key regulatory proteins has been implicated as one of the primary determinants of their stability, and thus, their availability, as was shown for cyclins, p53 and IkB. Upon identifying Ub in the fraction which mediates transcriptional inhibitor activities he has studied c-jun ubqt via an in vitro system which enabled one to determine that c-jun ubqt is targeted, in part, by jun-N-kinase (JNK), a multimember family of stress-activated protein kinases which also phosphorylate c-jun on the N-terminal region. It is his working hypothesis that JNK-mediated c-jun ubqt is a key upstream regulatory event which dictates stability of c-jun and potentially of other JNK substrates and, in turn, the fate of the UV-response and UV-mediated tumorigenesis. To test this hypothesis he proposes to identify mechanisms involved in JNK-mediated ubqt of jun; to determine the functional significance consequences of such changes to the biology of melanoma. Specifically, he proposes to: 1) Apply the in vitro model toward an in vivo controlled ubqt system to evaluate and compare the effects of JNK on ubqt/degradation of c-jun in melanoma vs. Fibroblasts settings. 2) Determine the biological significance of JNK-mediated ubqt in the cellular stress response. 3) Identified peptides and regions on JNK that modulate JNK-mediated ubqt. 4) determine how modulation of JNK-targeted c-jun ubqt will affect characteristic melanoma phenotypes by using both primary melanoma and its derived metastatic form as a relevant model. 5) Determine whether JNK can also target ubqt of other substrates known to play an important role in the UV-response, including ATF2 and p53. In all, the proposed studies will explore one of the far upstream regulatory components involved in c-jun stability and thus, determine its availability. Understanding of this key regulatory process will enable new innovative design of measures towards regulation of transcription factors important to the biology of melanoma.
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