PHOSPHORYLATION OF COMPLEMENT COMPONENT C3 AFTER SYNTHESIS IN U937 CELLS BY A PUTATIVE PROTEIN-KINASE, CASEIN KINASE 2, WHICH IS REGULATED BY CD11B - EVIDENCE THAT MEMBRANE-BOUND PROTEASES PREFERENTIALLY CLEAVE PHOSPHORYLATED C3

It was our aim in this study to investigate the possibility that the t hird component of complement (C3) is phosphorylated during synthesis a nd secretion in U937 cells. Labelling of U937 cells with [P-32]P-i, fo llowed by immunoprecipitation of C3 from cell lysates and culture supe rnatants at different time points, showed that C3 was phosphorylated i ntracellularly immediately before release into the medium, which initi ated cleavage of the protein into an iC3b-like fragment. Stimulation o f CD11b/CD18 increased phosphorylation 7-fold, from a basal level of 2 %. The phosphorylation sites in C3 did not resemble those described pr eviously for casein kinase (CK) 1, cAMP-dependent protein kinase A or calcium-and phospholipid-dependent protein kinase C. Instead, protein kinase CK2 was suggested inasmuch as: (1) CK2 was detected both on the cell surface and on shed microparticles; (2) phosphorylation of purif ied C3 by microparticles was abolished by a monoclonal antibody, anti- CK2; (3) the [P-32]P-i tag of both phosphorylated C3 (secreted from U9 37 cells) and of microparticle-phosphorylated C3, which was cleaved ei ther by membrane proteases or by leucocyte elastase, was found in a 40 and a 70 kDa polypeptide; (4) both secreted C3 and C3 phosphorylated in vitro were much more susceptible to cleavage by proteases. Generati on of C3 fragments provides a means by which U937 cells can stimulate nearby cells which are expressing complement receptors. The present st udy demonstrates that the cleavage of C3 is controlled by an intracell ular phosphorylation event regulated by CD11b/CD18.