IDENTIFICATION OF A CRYPTIC PROTEIN-KINASE CK2 PHOSPHORYLATION SITE IN HUMAN-COMPLEMENT PROTEASE CLR, AND ITS USE TO PROBE INTRAMOLECULAR INTERACTION

Treatment of human C(1) over barr$ by CK2 resulted in the incorporatio n of [P-32]phosphate into the N-terminal alpha region of its non-catal ytic A chain. Fragmentation of P-32-labelled C(1) over barr$ followed by N-terminal sequence and mass spectrometry analyses allowed identifi cation of Ser(189) as the phosphorylation site, Accessibility of Ser(1 89) was low in intact C1r, due in part to the presence of one of the o ligosaccharides borne by the a region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r- C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was e nhanced in the isolated alpha fragment and insensitive to calcium, Tak en together, these data provide support for the occurrence of a Ca2+-d ependent interaction between the alpha region and the remainder of the C1r molecule.