Expression and regulation of phospholipase D isoenzymes in human melanoma cells and primary melanocytes.
Phospholipase D (PLD) is a highly regulated enzyme involved in lipid-mediated signal transduction processes affecting vesicular trafficking and cytoskeletal reorganization. It is regulated by protein kinase C, adenosine diphosphate (ADP)-ribosylation factors and Rho family proteins, and both protein kinase C and Rho family proteins have been implicated in the metastatic potential of melanoma. We analysed PLD in four human melanoma cell lines and in primary human melanocytes. Melanoma cell lines showed phosphatidylcholine-hydrolysing, phosphatidylinositol 4,5-bisphosphate-dependent PLD activity, which was activated by phorbol ester and a non-hydrolysable guanosine triphosphate (GTP) analogue in a dose-dependent and synergistic manner, whereas primary melanocytes exhibited only low PLD activity compared with the melanoma cell lines. As determined by reverse transcription polymerase chain reaction, both splicing variants of PLD1, PLD1a and PLD1b, and the isoenzyme PLD2, are expressed in melanoma cells and melanocytes. Western blot analysis showed that PLD1 expression was low in primary melanocytes in contrast to melanoma cells, which is in agreement with our finding of low activity. Interestingly, Rho protein mRNA was elevated in all melanoma cell lines. We conclude that in human melanoma cells, the PLD activity that is stimulated by phorbol ester requires ADP-ribosylation factor, protein kinase C and Rho proteins for full activity, and most probably represents the isoenzyme PLD1.
Riebeling
Christian
C
Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Germany.
Müller
Carola
C
Geilen
Christoph C
CC
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D017398:Alternative Splicing; D015153:Blotting, Western; D002455:Cell Division; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D009362:Neoplasm Metastasis; D019301:Oleic Acid / Q000378:metabolism; D010703:Phorbol Esters; D010739:Phospholipase D / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D018076:DNA, Complementary; D010703:Phorbol Esters; D020033:Protein Isoforms; D012333:RNA, Messenger; D019301:Oleic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D012313:RNA; D006160:Guanosine Triphosphate; D011493:Protein Kinase C; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D020727:ADP-Ribosylation Factors; D020741:rho GTP-Binding Proteins