Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity

William D. Singer, H. Alex Brown, Xuejun Jiang, Paul C. Sternweis

Research output: Contribution to journalArticle

230 Scopus citations

Abstract

Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP-ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide-independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol Chem, 270, 14944-14950). Here, we report purification of the latter factor, its identification as the α isoform of protein kinase C (PKCα), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCα or recombinant PKCα (rPKCα) occurred in the absence of any nueleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKCα with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCα without affecting activation of PLD. Finally, gel filtration of PKCα that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation.

Original languageEnglish (US)
Pages (from-to)4504-4510
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number8
DOIs
StatePublished - Feb 23 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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