Phosphorylation of human fibrinogen in vitro with protein kinase C: characterization of the phosphorylated sites.
Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D004586:Electrophoresis; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013154:Spleen / Q000201:enzymology; D013552:Swine; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D010710:Phosphates; D012694:Serine; D005340:Fibrinogen; D011493:Protein Kinase C; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide