Requirement of Syk-phospholipase C-gamma2 pathway for phorbol ester-induced phospholipase D activation in DT40 cells.
BACKGROUND
Treatment of many cell types with phorbol esters stimulates phospholipase D (PLD) activity implying regulation of the enzyme by protein kinase C. Studies of the effects of several protein-tyrosine kinase (PTK) inhibitors have suggested that PTK(s) play some roles in the phorbol ester-induced PLD activation, but it remains unclear how and which PTK(s) is involved in this pathway. In this study, we investigated the roles of Syk and other PTKs for the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced PLD activation in K562 and DT40 cells.


RESULTS
TPA-induced PLD activation was remarkably reduced in both Syk dominant negative mutant K562 cells and Syk deficient DT40 B cells. Mutational analysis further indicated that two major autophosphorylation sites (Tyr-518 and Tyr-519) of Syk are critical for PLD activation. Similarly, TPA-induced 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 activation, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was remarkably reduced.


CONCLUSIONS
We demonstrated that the Syk, Btk and PLC-gamma2 pathways are required for TPA-induced PLD activation in DT40 cells.
Hitomi
T
T
Department of Biochemistry Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
Yanagi
S
S
Inatome
R
R
Ding
J
J
Takano
T
T
Yamamura
H
H
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D020014:K562 Cells; D009154:Mutation; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D000072377:Syk Kinase; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011947:Receptors, Antigen, B-Cell; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate