TY - JOUR
T1 - Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity
AU - Singer, William D.
AU - Brown, H. Alex
AU - Jiang, Xuejun
AU - Sternweis, Paul C.
PY - 1996/2/23
Y1 - 1996/2/23
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.271.8.4504
DO - 10.1074/jbc.271.8.4504
M3 - Article
C2 - 8626805
AN - SCOPUS:0029671455
SN - 0021-9258
VL - 271
SP - 4504
EP - 4510
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 8
ER -