Study of the role of protein kinase C-phosphoipase D signaling pathway in pigment cells.

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It is well known that phospholipase D (PLD) plays a crucial role in the signal transduction of many types of cells, and is activated by protein kinase C α (PKCα) when cells are stimulated. To elucidate the role of PLD in melanoma, the expression of PLD1 and PKCα in primary and metastatic lesions of acral lentiginous melanoma (ALM) and superficial spreading melanoma (SSM) was investigated using immunohistological techniques. In addition, the mechanism of regulation of PLD1 by PKCα was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both PLD1 and DKCα were strongly expressed in primary and metastatic lesions of SSM. Conversely, in ALM lesions, the expression of these two proteins increased dramatically with tumor progression ; the expression of both PLD1 and PKCα was almost negative in the radial growth phase of primary ALM lesions, and increased synchronously in a progression-related manner in advanced ALM esions, including vertical gro … More wth phase and metastatic lesions. Immunoprecipitation study showed that PLD1 and PKCα are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of PLD1 and PKCα, or PLD1 and the kinase-negative mutant of PKCα revealed that both PKCα and the kinase-negative mutant of PKCα are associated with PLD1 in melanoma cells in the absence of an extemal signal. Overexpression of PKCα or the kinase-negative mutant of PKCα in melanoma cells by the adenovirus vectors resulted in the enhancement of basal PLD activity in a viral dose-dependent manner. Furthermore, enhanced basal PLD activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of PLD1 and PKCα plays a rote in the progression of ALM from the radial growth phase to the vertical growth phase. The present results also suggest that PKCα associates with PLD1 and enhances basal PLD activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential. Less