It is becoming clear that growth factors relay mitogenic and developmental signals from the cell surface into the cytoplasm and nucleus. Our long term goal is to understand the biochemical mechanisms by which these signals translate into melanocyte-specific gene expression. Our previous work has shown that normal melanocytes depend on synergistic mitogens in the extracellular environment for proliferation and expression of differentiated functions. The synergistic mitogens regulate melanocyte morphology and pigmentation. Our hypothesis is that growth factors activate pre-existing transcription factors which in turn upregulate MITF (microphthalmia associated transcription factor, the human homologue of mouse mi), a recently discovered transcription factor implicated in melanocytic functions. We already identified at least part of the intermediates activated by synergistic growth factors, i.e., the MAPK cascade (mitogen-activated protein kinase), the serine/threonine ribosomal S6 kinases p70S6K and p90RSK, and the transcription factor CREB(cyclic AMP/Ca++-responsive element binding protein). We identified p90RSK as the novel CREB-Ser133 kinase that phosphorylates CREB in vitro and we showed that ectopic expression of an unphosphorylatable CREB mutant suppresses pigmentation. This proposal focuses on further elucidation of the role of mediators in signal transduction in regulating melanocyte proliferation/differentiation programs. The Three Specific Aims are: 1) To confirm that the Ribosomal S6 Kinase p90RSK activates CREB in vivo. This goal will be pursued by: a) interfering with the activity of this kinase through transfection of melanocytes with antisense oligonucleotides directed against RSK as well as transfections with recombinant genes encoding dominantly-interfering MAPK (the upstream regulator of RSK) or p90RSK products; and b) elevate specific kinases by transfection with recombinant genes encoding constitutively active enzymes. 2) To determine the molecular mechanism by which a dominantly negative unphosphorylatable CREB (CREB M1, in which Ser133 is substituted with Ala) suppresses pigmentation and proliferation., We will explore the possibilities that CREB M1 acts as a suppressor of melanocyte specific gene expression directly, and/or indirectly, through regulation of Fos and mi. 3) To study the relationship between aberrant expression of bFGF, the activity,of bFGFR kinase and expression of MITF. This goal will be accomplished by transfecting melanoma cells, with eukaryotic expression vectors encoding a) anti-sense bFGF mRNA; b) a kinase-truncated, dominant negatively acting bFGF receptor (FGFR1/FLG); and c) an inducible mi minigene.
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