Abstract
The phosphorylation of human C3a (hC3a, anaphylatoxin) by two distinct protein kinases (PKA and CK-I) and the effect of cholesterol-3-sulfate (CH-3S) on this phosphorylation were biochemically investigated in vitro. It was found that (i) hC3a functions as a phosphate acceptor for PKA and CK-I, but not for CK-II; (ii) the CK-I-mediated phosphorylation of hC3a requires the presence of 3 μM CH-3S in a manner similar to the phosphorylation of HMG1 (CH-3S-binding protein) by CK-I; and (iii) CH-3S inhibits the PKA-mediated phosphorylation of hC3a in a dose-dependent manner (ID50=approx. 2 μM). As expected, hC3a containing high levels of Arg- and Lys-residues stimulated approx. 3-fold CK-II activity (phosphorylation of α-casein) in vitro. However, no significant effect of hC3a on CK-II activity was observed when hC3a was preincubated with CH-3S or fully phosphorylated by PKA in vitro. Furthermore, preincubation of hC3a with CH-3S diminished the ability of hC3a to induce vascular permeability in rats. The results provided here suggest that (i) hC3a is a CH-3S-binding protein; and (ii) CH-3S functions as a potent inhibitor for its physiological activities, including phosphorylation by PKA and CK-I, in vitro.
