Understanding mechanisms underlying the integration of signal transduction pathways remains among the challenging tasks in dissection of diverse cellular regulatory events. Studies supported by this grant led to the discovery that RACK1 mediates JNK activation by PKC, which is required for activation of JNK by diverse stimuli. Our finding provides a functional link between PKC and JNK signaling cascades. Since RACK1 is also implicated in diverse cellular functions, including cell adhesion and mRNA translation, it is our hypothesis that the PKC-RACK1 module is a central regulator of JNK activities in the cellular response to stress, including cell contact and mRNA translation. To test this possibility we will: 1. Determine the contribution of RACKI to JNK-dependent regulation of cell adhesion and motility. We will assess the role of JNK in RACK1-dependent changes in localization and phosphorylation of cytoskeletal proteins involved in cell adhesion and motility. (2) Assess the role of the PKC-RACK1-JNK pathway in the regulation of mRNA translation. Studies in S. pombe and vertebrates support the hypothesis of RACK1's role in regulating mRNA translation. In collaboration with Nahum Sonenberg we will directly assess the role of the PKC-RACK1-JNK pathway in regulating selected (as well as general) mRNA translation. (3). Explore feed- forward mechanism in RACK1 activation of JNK. Our preliminary results reveal that c-Jun and Stat3 regulate the transcription of RACK1, PDK1 (the upstream kinase for PKC), suggesting a feed-forward loop mechanism. Cells subjected to physiological stimuli that induce PKC and JNK will be monitored for changes in (a) duration and level of JNK activation (b) transcriptional regulation of RACK1 and PDK1 by Stat3 and c-Jun (c) the degree and kinetics of PDK1 and PKC activities (4). Characterize changes in the RACK-JNK pathway in melanomas. Our preliminary results indicate that the RACK1-JNK pathway is modified in melanomas, in part due to the effect of ERK on c-Jun expression. These prompt us to test the hypothesis that upregulated MAPK (which is seen in most melanomas due to B-Raf or N-Ras mutation) impact RACK1- JNK pathway. Thus, we will determine mechanism underlying the link between MAPK and c-Jun and the RACK1 pathway, target the key components (ERK, c-Jun, RACK1) and monitor changes in the biology of melanomas, and assess possible correlation between ERK, JNKSer129, PKC and tumor prognosis using melanoma tissue arrays. Our studies are expected to provide new important insight into the cross talk between JNK and PKC signaling and their implications for diverse cellular processes important in cell growth and motility which are altered in human melanomas.
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