In vitro 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 (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm P1-protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion P1-protamine, and most probably the homologous Thr-43 in bull P1-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation 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.
Pirhonen
A
A
Department of Biochemistry & Biotechnology, University of Kuopio, Finland.
Valtonen
P
P
Linnala-Kankkunen
A
A
Mäenpää
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006736:Horses; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011479:Protamines / Q000737:chemistry / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013045:Species Specificity; D013094:Spermatozoa / Q000378:metabolism
D011479:Protamines; C046533:protamine P1; D011494:Protein Kinases; D011493:Protein Kinase C