The effects of cholesterol-3-sulfate (CH-3S) on the phosphorylation of human C3a (hC3a) in vitro and on the ability of hC3a to induce vascular permeability in rats.
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 microM 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=approximately 2 microM). As expected, hC3a containing high levels of Arg- and Lys-residues stimulated approx. 3-fold CK-II activity (phosphorylation of alpha-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.
Laboratory of Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, Kitasato, Sagamihara, Japan.
Fumitaka
F
Kawakami
Masaki
M
Ito
Yuya
Y
Matsuda
Izumi
I
Hayashi
Kenzo
K
Ohtsuki
D000818:Animals; D002199:Capillary Permeability; D047389:Casein Kinase I / Q000378:metabolism; D002788:Cholesterol Esters / Q000737:chemistry / Q000494:pharmacology*; D015926:Complement C3a / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012867:Skin / Q000098:blood supply*
D002788:Cholesterol Esters; D015926:Complement C3a; D010770:Phosphotransferases; D047389:Casein Kinase I; D017868:Cyclic AMP-Dependent Protein Kinases; C007045:cholesteryl sulfate