Vol. 189, No. 1, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS November 30, 1992 Pages 40-46 IMMIJNOCYTOCHKMICAL EXPRESSION AND LOCALIZATION OF PROTEIN KINASE C IN BOVINE AORTIC ENDOTHELIAL CELLS1 Oscar R. Resales*, Carlos Isales*,MichaelNathanson*, and Bauer E. Sumpio+ Departments of *Medicine and *Surgery, Yale University School of Medicine, New Haven, CT 06510 Received October 1, 1992 Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cytosolic fraction (66%)in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the a and B PKC isoforms. y PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC a immunofluorescence redistributed to the perinuclear region whereas PKC B staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation. 0 1992 Rcademlc Press, Inc. Protein kinase C (PKC) is a group of calcium- and phospholipid-dependent protein kinases which appears to be involved in the signal transduction response of many cell systems to a variety of hormones, growth factors and drugs (1). Cloning studies have demonstrated that PKC is a family of closely related genes that encode a number of isoenzymes which exhibit distinct tissue-specific patterns of expression. Initially, three different genes (a, B, and y) were reported; additional cDNA clones (a, a and 0 have subsequently been isolated (2) and others will probably be discovered in the future. Activation of PKC is thought to occur in response to the synergistic action of diacylglycerol and calcium, both of which can be generated by signal-induced hydrolysis of membrane phospholipids. PKC can be measured in both cytosolic and particulate fractions. An indication of the activation of PKC is an increase in membrane-associated PKC activity following cell stimulation (2). This "translocation" has been noted in a variety of cell types including cultured BAEC (3). However, the individual patterns of expression of the PKC isoenzymes in BAEC have not been described. 1Supported by grants to B.E.S. from the NIH (HL40305, HL47345), and the Veterans Administration. O.R.R. was an AHA Research Fellow, Connecticut Affiliate. 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 40