Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils - Enhancement of Fgr activity

In human neutrophils, the activation of phospholipase D and the Tyr phospho rylation of proteins are early signaling events upon cell stimulation. We f ound that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the exp ense of phosphatidic acid (PA), decreased the phorbol myristate acetate-sti mulated Tyr phosphorylation of endogenous proteins (42, 115 kDa), When neut rophil cytosol was incubated in the presence or absence of PA, these and ot her endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphor ylation in the cell-free assay. Similarly, other phospholipids (phosphatidy lcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserin e, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate ) showed little ability to stimulate Tyr phosphorylation, These data sugges t that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a pe ak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-1 5578). Among the protein Tyr kinases expressed in neutrophils, only Fgr elu ted exclusively in the peak of PA-dependent Tyr phosphorylating activity, I mportantly, Fgr isolated from unstimulated neutrophil. lysates showed incre ased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in ce lls stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytoc halasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.