title: Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha.
abstract: We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-gammaS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-alpha activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-alpha is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.
authors.affiliation: Department of Pharmacological Sciences, Stony Brook Health Sciences Center, Stony Brook, New York 11794-8651, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Hammond
authors.forename: J M
authors.initials: JM
authors.lastname: Jenco
authors.forename: S
authors.initials: S
authors.lastname: Nakashima
authors.forename: K
authors.initials: K
authors.lastname: Cadwallader
authors.forename: Q
authors.initials: Q
authors.lastname: Gu
authors.forename: S
authors.initials: S
authors.lastname: Cook
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
authors.forename: M A
authors.initials: MA
authors.lastname: Frohman
authors.forename: A J
authors.initials: AJ
authors.lastname: Morris
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D017398:Alternative Splicing; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha
chemical_list: D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D000246:Adenosine Diphosphate Ribose; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium