Regulation of phospholipase D2 activity by protein kinase C alpha.
It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, Gö 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.
Chen
Jun-Song
JS
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Exton
John H
JH
D000818:Animals; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D010739:Phospholipase D / Q000096:biosynthesis*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D012694:Serine / Q000737:chemistry; D013347:Subcellular Fractions; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C105606:phospholipase D2; D010739:Phospholipase D; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220