Regulation of Melanogenesis at the Sub-Cellular Level in Cloudman Murine Melanoma Cells

Kenneth E. Rosenzweig, Yale University


The decarboxylation of dopachrome to 5,6-dihydroxyindole (DHI) appears to be a major control point in the biosynthesis of melanin, in particular the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The recent discovery of a factor, DHICA stablin, that stabilizes DHICA and inhibits its conversion to DHI has added insight into the regulation of this intermediary compound. This study has shown that DHICA stablin activity is present in the melanosomal fraction of Cloudman murine melanoma cells and that this activity was observed by a new method using two complementary decarboxylase assays. When three known decarboxylase stabilizing cofactors (biotin, pyridoxal phosphate, and pyruvate) were added to melanosomal extracts, DHICA decarboxylase activity was enhanced but these factors did not decrease the lability of the decarboxylase enzyme. Protein kinases have been shown to mediate an adenylate cyclase system that is involved in the regulation of morphology and proliferation of Cloudman murine melanoma cells. It is also known that coated vesicles are involved in the transfer of protein kinases and melanin precursors between internal cell components, which raises the possibility that coated vesicles are involved in the initiation of melanin synthesis and the formation of melanosomes. In order to further characterize this process, coated vesicles were isolated and the activity of their associated protein kinases was examined in the presence of calcium, magnesium, EGTA (a known calcium chelator) and calmodulin (a ubiquitous intracellular receptor for calcium that mediates most calcium regulated processes). Protein kinase activity associated with coated vesicles isolated from Cloudman murine melanoma cells was shown to be stimulated by pre incubation with melanocyte stimulating hormone. This protein kinase appeared to be magnesium dependent and inhibited by calcium. These results were demonstrated by an assay of protein kinase activity as well as autoradiography. Together, these studies reveal new insights into the sub cellular regulation of melanogenesis, and they underscore the high degree of complexity involved. 2