Characterization of a rat brain phospholipase D isozyme.
We have recently cloned a cDNA encoding a phospholipase D (PLD) from rat brain and named it rPLD1. It shows 90% amino acid identity with the human PLD isoform hPLD1b. We have expressed rPLD1 as a histidine-tagged fusion protein in insect (Sf9) cells using the expression vector pBlueBacHis and purified the recombinant protein to homogeneity by Ni2+-agarose affinity chromatography. Phosphatidylinositol 4,5-P2 and phosphatidylinositol 3,4,5-P3 activated the PLD equipotently, but other acidic phospholipids were ineffective. The activity of rPLD1 was dependent on both Mg2+ and Ca2+. It was specific for phosphatidylcholine and showed a broad dependence on pH with optimum activity at pH 6.5-7.5. The enzyme was inhibited by oleate and activated by the small G proteins ARF3 and RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Protein kinase C (PKC)-alpha and -betaII, but not PKC-gamma, -delta, -epsilon, or -zeta, activated rPLD1 in a manner that was stimulated by phorbol ester but did not require ATP. Neither synergistic interactions between ARF3 and RhoA nor between these G proteins and PKC-alpha or -betaII were observed. Recombinant PKC-alpha and -betaII phosphorylated purified rPLD1 to high stoichiometry in vitro, and the phosphorylated PLD exhibited a mobility shift upon electrophoresis. Phosphorylation of the PLD by PKC was correlated with inhibition of its catalytic activity. rPLD1 bound to concanavalin A-Sepharose beads, and its electrophoretic mobility was altered by treatment with endoglycosidase F. The amount of PLD bound to the beads was decreased in a concentration-dependent manner when tunicamycin was added to the Sf9 expression system. Tunicamycin also decreased membrane localization of rPLD1. These results suggest that rPLD1 is a glycosylated protein and that it is negatively regulated by phosphorylation by PKC in vitro.
Min
D S
DS
Department of Molecular Physiology and Biophysics, and Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Park
S K
SK
Exton
J H
JH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D019301:Oleic Acid / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity
D007527:Isoenzymes; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D019301:Oleic Acid; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium