Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: enhancement of Fgr activity.
In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other 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 phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak 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, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.
Sergeant
S
S
Departments of Biochemistry and Medicine, Division of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. ssergean@wfubmc.edu
Waite
K A
KA
Heravi
J
J
McPhail
L C
LC
D020001:1-Butanol / Q000494:pharmacology; D002478:Cells, Cultured; D000431:Ethanol / Q000494:pharmacology; D020404:Glycerophospholipids / Q000494:pharmacology; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000494:pharmacology / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019061:src-Family Kinases
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D000431:Ethanol; D020001:1-Butanol; D011505:Protein-Tyrosine Kinases; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate