TY - JOUR
T1 - Characterization of a rat brain phospholipase D isozyme
AU - Min, Do Sik
AU - Park, Seung Kiel
AU - Exton, John H.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1998/3/20
Y1 - 1998/3/20
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.273.12.7044
DO - 10.1074/jbc.273.12.7044
M3 - Article
C2 - 9507013
AN - SCOPUS:0000578505
VL - 273
SP - 7044
EP - 7051
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 12
ER -