Protein kinase C α associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in human melanoma cells

It is well known that phospholipase D plays a crucial part in the signal transduction of many types of cells, and is activated by protein kinase C alpha when cells are stimulated. To elucidate the role of phospholipase D in melanoma, the expression of phospholipase D-1 and protein kinase C alpha in primary and metastatic lesions of acral lentiginous melanoma and superficial spreading melanoma was investigated using immunohistologic techniques. In addition, the mechanism of regulation of phospholipase D-1 by protein kinase C alpha was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both phospholipase D-1 and protein kinase C alpha were strongly expressed in primary and metastatic lesions of superficial spreading melanoma. Conversely, in acral lentiginous melanoma lesions, the expression of these two proteins increased dramatically with tumor progression; the expression of both phospholipase D-1 and protein kinase C alpha was almost negative in the radial growth phase of primary acral lentiginous melanoma lesions, and increased synchronously in a progression-related manner in advanced acral lentiginous melanoma lesions, including vertical growth phase and metastatic lesions. Immunoprecipitation study showed that phospholipase D-1 and protein kinase C alpha are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of phospholipase D-1 and protein kinase C alpha, or phospholipase D-1 and the kinase-negative mutant of protein kinase C alpha revealed that both protein kinase C alpha and the kinase-negative mutant of protein kinase C alpha are associated with phospholipase D-1 in melanoma cells in the absence of an external signal. Overexpression of protein kinase C alpha or the kinase-negative mutant of protein kinase C alpha in melanoma cells by the adenovirus vectors resulted in the enhancement of basal phospholipase D activity in a viral concentration-dependent manner. Furthermore, enhanced basal phospholipase D activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of phospholipase D-1 and protein kinase C alpha plays a part in the progression of acral lentiginous melanoma from the radial growth phase to the vertical growth phase. The present results also suggest that protein kinase C alpha associates with phospholipase D-1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential.