SUBCELLULAR-DISTRIBUTION OF PROTEIN-KINASE C-ALPHA AND BETA-I IN BOVINE SPERMATOZOA, AND THEIR REGULATION BY CALCIUM AND PHORBOL ESTERS

Protein kinase C (PKC), the major cell target for tumor-promoting phor bol esters, is central to many signal transduction pathways. Previousl y we have demonstrated the presence of PKC in ram and bovine spermatoz oa. However, the relative distribution of various PKC isozymes in the cytosolic and membrane fractions and their regulation by calcium and p horbol esters have not been elucidated. Immunocytochemical studies and Western blotting with antibodies specific for individual isoforms rev ealed that at least two PKC isoforms, cPKC(alpha) and cPKC(beta)I, are found in bovine sperm cells. We demonstrate, by Western blotting anal ysis, that both PKC isozymes were predominantly localized in the cytos ol when subcellular fractionation was carried out in the presence of E GTA. When cell lysis was carried out in the presence of Ca2+, most PKC alpha and PKCbetaI redistributed to the particulate fraction, Treatmen t of sperm cells with the biologically active phorbol ester 12-O-tetra decanoyl phorbol-13-acetate (TPA) resulted in a rapid and extensive tr anslocation of cytosolic PKCalpha and cytosolic PKCbetaI to the membra ne fraction within 1 min. Furthermore, PKC's total activity was measur ed as a calcium- and phospholipid-dependent phosphorylation of a synth etic peptide in the cytosolic and membrane fractions derived from cont rol and TPA-treated spermatozoa. TPA evoked a decrease in cytosolic PK C activity, accompanied by an increase in the activity associated with the plasma membrane fraction. This translocation of PKC enzymes may e nsure their binding to intracellular receptor proteins (''RACKs'') and the phosphorylation of specific substrates, which appears to determin e their physiological function. The presence of RACK in the membrane f raction of bovine sperm cells was confirmed with use of an antibody di rected against the RACK protein, Previously we demonstrated the involv ement of PKC in sperm acrosomal exocytosis, a process induced by signa l transduction events. Thus, our results suggest that the rapid associ ation of PKCalpha and PKCbetaI with the sperm plasma membrane, as show n in the present work for the first time, may be an early event in spe rm cell regulation, leading to acrosomal exocytosis and fertilization.