In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.
Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.
Nearly half the patients first identified their early DFSP-related skin changes as patches. Both this frequency and the long duration at this preprotuberant stage should prompt dermatologists to consider the diagnosis of DFSP earlier, in order to make surgical treatment easier.
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