P2 PROTAMINES ARE PHOSPHORYLATED IN-VITRO BY PROTEIN-KINASE-C, WHEREAS P1 PROTAMINES PREFER CAMP-DEPENDENT PROTEIN-KINASE - A COMPARATIVE-STUDY OF 5 MAMMALIAN-SPECIES

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro pho sphorylation reactions using cAMP-dependent protein kinase (PKA) and p rotein kinase C (PKC). All P1 protamines were phosphorylated by PKA, w hereas P2 protamines were phosphorylated only by PKC. In addition, hum an, stallion and boar, but not bull and ram, PZ protamines were phosph orylated by PKC. After phosphoamino acid analysis, the protamines show ing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and h uman (HP1) P1 protamines contained P-Ser after PKA phosphorylation, lo cated in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P- Thr, which could not be further localized in the sequence with the pre sent methods. After PKC phosphorylation, the internally located Ser re sidues in human (Ser21) and stallion (Ser29) P1 protamines were phosph orylated and, in boar P1 protamine, only Thr43 was slightly phosphoryl ated. The N-terminally located Ser residues in P1 protamines, which ar e known to be phosphorylated in vivo, were not phosphorylated by eithe r kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was t he main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamine s.