Subcellular Distribution of Protein Kinase Cα and β1 in Bovine Spermatozoa, and their Regulation by Calcium and Phorbol Esters1

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, is central to many signal transduction pathways. Previously we have demonstrated the presence of PKC in ram and bovine spermatozoa. However, the relative distribution of various PKC isozymes in the cytosolic and membrane fractions and their regulation by calcium and phorbol esters have not been elucidated. Immunocytochemical studies and Western blotting with antibodies specific for individual isoforms revealed that at least two PKC isoforms, cPKC alpha and cPKC betaI, are found in bovine sperm cells. We demonstrate, by Western blotting analysis, that both PKC isozymes were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of EGTA. When cell lysis was carried out in the presence of Ca2+, most PKC alpha and PKC betaI redistributed to the particulate fraction. Treatment of sperm cells with the biologically active phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) resulted in a rapid and extensive translocation of cytosolic PKC alpha and cytosolic PKC betaI to the membrane fraction within 1 min. Furthermore, PKC's total activity was measured as a calcium- and phospholipid-dependent phosphorylation of a synthetic peptide in the cytosolic and membrane fractions derived from control and TPA-treated spermatozoa. TPA evoked a decrease in cytosolic PKC activity, accompanied by an increase in the activity associated with the plasma membrane fraction. This translocation of PKC enzymes may ensure their binding to intracellular receptor proteins ("RACKs") and the phosphorylation of specific substrates, which appears to determine their physiological function. The presence of RACK in the membrane fraction of bovine sperm cells was confirmed with use of an antibody directed against the RACK protein. Previously we demonstrated the involvement of PKC in sperm acrosomal exocytosis, a process induced by signal transduction events. Thus, our results suggest that the rapid association of PKC alpha and PKC betaI with the sperm plasma membrane, as shown in the present work for the first time, may be an early event in sperm cell regulation, leading to acrosomal exocytosis and fertilization.