Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes.
Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.
Markou
Thomais
T
National Heart and Lung Institute Division, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom.
Yong
Chee Shin
CS
Sugden
Peter H
PH
Clerk
Angela
A
D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000302:isolation & purification; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002867:Chromones / Q000494:pharmacology; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011233:Precipitin Tests; D051745:Protein Kinase C-delta / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors
C412373:AG 1879; D002457:Cell Extracts; D002867:Chromones; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D009025:Morpholines; D010982:Platelet-Derived Growth Factor; D011743:Pyrimidines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051745:Protein Kinase C-delta; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate