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-α

Scott M. Hammond, John M. Jenco, Shigeru Nakashima, Karen Cadwallader, Qu Ming Gu, Simon Cook, Yoshinori Nozawa, Glenn D. Prestwich, Michael A. Frohman, Andrew J. Morris

Research output: Contribution to journalArticlepeer-review

504 Scopus citations

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 585624) 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-γS-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-α 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-α 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.

Original languageEnglish
Pages (from-to)3860-3868
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number6
DOIs
StatePublished - 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of '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-α'. Together they form a unique fingerprint.

Cite this