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J. Biol. Chem., Vol. 281, Issue 13, 8321-8331, March 31, 2006

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Regulation of Protein Kinase C by Phorbol Ester, Endothelin-1, and Platelet-derived Growth Factor in Cardiac Myocytes^*

From the National Heart and Lung Institute Division, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom

Protein kinase C (PKC) 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" PKC for activation, it could be regulatory. We examined the regulation of PKC in cardiac myocytes by endothelin-1 (G_q 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 PKC(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKC to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKC subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, 25% of PKC 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 PKC activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKC 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 PKC and the increase in phosphatidylserine/PMA-independent activity persisted after PKC retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKC becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.

Received for publication, August 1, 2005 , and in revised form, December 13, 2005.

Note Added in Proof—While this manuscript was under revision, Rybin and Steinberg (Rybin, V. O., and Steinberg, S. F. (2005) Am. J. Physiol. Cell Physiol. Epub ahead of print, October 19) also noted that phosphorylation of PKC may reduce its immunoprecipitation with the BD Bioscience antibody, as we have shown here (Fig. 7). Although we suggested that this may result from Tyr-phosphorylation of PKC, Rybin and Steinberg attribute this to an event that can be inhibited by GF109203X (i.e. it may be caused by Ser-/Thr-phosphorylation).

^* This work was supported by British Heart Foundation Grant RG2001017. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: National Heart and Lung Institute Division, Faculty of Medicine, Imperial College London, Flowers Bldg. (4th Floor), Armstrong Rd., London SW7 2AZ, UK. Tel.: 44-20-7594-3410; Fax: 44-20-7594-3419; E-mail: p.sugden{at}imperial.ac.uk. ² To whom correspondence may be addressed. Tel.: 44-20-7594-3009; Fax: 44-20-7594-3419; E-mail: a.clerk{at}imperial.ac.uk.

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