Requirement of Syk-phospholipase C-gamma 2 pathway for phorbol ester-induced phospholipase D activation in DT40 cells

Background: Treatment of many cell types with phorbol esters stimulates pho spholipase D (PLD) activity implying regulation of the enzyme by protein ki nase C. Studies of the effects of several protein-tyrosine kinase (PTK) inh ibitors have suggested that PTK(s) play some roles in the phorbol ester-ind uced PLD activation, but it remains unclear how and which PTK(s) is involve d in this pathway. In this study, we investigated the roles of Syk and othe r PTKs for the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA)-i nduced PLD activation in K562 and DT40 cells. Results: TPA-induced PLD activation was remarkably reduced in both Syk domi nant negative mutant K562 cells and Syk deficient DT40 B cells. Mutational analysis further indicated that two major autophosphorylation sites (Tyr-51 8 and Tyr-519) of Syk are critical for PLD activation. Similarly, TPA-induc ed PLD activation was reduced in Btk deficient cells, but unaffected in Lyn deficient cells. Finally, in cells deficient in the PLC-gamma2, one of the phosphorylated substrates regulated by Syk and Btk, TPA-induced PLD activa tion, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis wa s remarkably reduced. Conclusions: We demonstrated that the Syk, Btk and PLC-gamma2 pathways are required for TPA-induced PLD activation in DT40 cells.