CHARACTERIZATION OF A RAT-BRAIN PHOSPHOLIPASE-D ISOZYME

We have recently cloned a cDNA encoding a phospholipase D (PLD) from r at brain and named it rPLD1, It shows 90% amino acid identity with the human PLD isoform hPLD1b, We have expressed rPLD1 as a histidine-tagg ed fusion protein in insect (Sf9) cells using the expression vector pB lueBacHis and purified the recombinant protein to homogeneity by Ni2+- agarose affinity chromatography. Phosphatidylinositol 4,5-P-2 and phos phatidylinositol 3,4,5-P-3 activated the PLD equipotently, but other a cidic phospholipids were ineffective. The activity of rPLD1 was depend ent 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 protei ns ARF3 and RhoA in the presence of guanosine 5'-3-O-(thio)triphosphat e. Protein kinase C (PKC)-alpha and -beta II, but not PKC-gamma, -delt a, -epsilon, or -zeta, activated rPLD1 in a manner that was stimulated by phorbol ester but did not require ATP, Neither synergistic interac tions between ARF3 and RhoA nor between these G proteins and PKC-alpha or -beta II were observed, Recombinant PKC-alpha and -beta II phospho rylated purified rPLD1 to high stoichiometry in vitro, and the phospho rylated PLD exhibited a mobility shift upon electrophoresis, Phosphory lation of the PLD by PKC was correlated with inhibition of its catalyt ic activity. rPLD1 bound to concanavalin A-Sepharose beads, and its el ectrophoretic mobility was altered by treatment with endoglycosidase F . The amount of PLD bound to the beads was decreased in a concentratio n-dependent manner when tunicamycin was added to the Sf9 expression sy stem, Tunicamycin also decreased membrane localization of rPLD1, These results suggest that rPLD1 is a glycosylated protein and that it is n egatively regulated by phosphorylation by PKC in vitro.