INVITRO PHOSPHORYLATION SITES OF STALLION AND BULL P1-PROTAMINES FOR CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE AND PROTEIN-KINASE-C

Fish and mammalian protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PK A) and protein kinase C (PKC), both requiring basic amino acids at the ir recognition sites, have previously been found to phosphorylate fish protamines in vitro. In this study, these enzymes were used to phosph orylate stallion and bull sperm P1-protamines in vitro. A species-spec ific difference was found, since PKA was able to phosphorylate both pr otamines while PKC phosphorylated only stallion protamine. Thr-4 1, th e only threonine residue in stallion Pl-protamine, and most probably t he homologous Thr-43 in bull P1-protamine are the sites for PKA phosph orylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by bo th kinases. Protamine phosphorylation by PKA was found to be almost in dependent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Ad dition of calcium, phosphatidylserine, and diolein caused a twofold st imulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian protamine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of P1-protamines.