Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils.
Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPgammaS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.
Kwak
J Y
JY
Department of Biochemistry, Dong-A University College of Medicine, Pusan, 602-103, Korea. jykwak@seunghak.donga.ac.kr
Uhlinger
D J
DJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009504:Neutrophils / Q000201:enzymology*; D010739:Phospholipase D / Q000097:blood*; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000097:blood
D046911:Macromolecular Substances; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010739:Phospholipase D; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate