title: Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.
abstract: Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.
authors.affiliation: Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
authors.forename: K N
authors.initials: KN
authors.lastname: Ekdahl
authors.forename: B
authors.initials: B
authors.lastname: Nilsson
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D003176:Complement C3 / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism; D017242:Complement Factor H / Q000378:metabolism; D017244:Complement Factor I / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D011494:Protein Kinases / Q000378:metabolism*; D005719:gamma-Globulins / Q000494:pharmacology
chemical_list: C493495:CFH protein, human; D003176:Complement C3; D010446:Peptide Fragments; D005719:gamma-Globulins; D003179:Complement C3b; D017242:Complement Factor H; D011494:Protein Kinases; D017244:Complement Factor I