Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity.
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 alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide 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 rPKC alpha 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 PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha 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.
Singer
W D
WD
Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Brown
H A
HA
Jiang
X
X
Sternweis
P C
PC
D020727:ADP-Ribosylation Factors; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006706:Homeostasis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D002352:Carrier Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium