Characterization of a rat brain phospholipase D isozyme

Do Sik Min, Seung Kiel Park, John H. Exton

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

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)-α and -βII, but not PKC- γ, -δ, -ε, or -ζ, 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-α or -βII were observed. Recombinant PKC-α, and -βII 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.

Original languageEnglish
Pages (from-to)7044-7051
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number12
DOIs
Publication statusPublished - 1998 Mar 20

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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