J Biol Chem, Vol. 273, Issue 7, 3901-3908, February 13, 1998

Cysteine Oxidation in the Mitogenic S100B Protein Leads to Changes in Phosphorylation by Catalytic CKII- Subunit

Christian Scotto, Yves Mély^§, Hiroshi Ohshima^¶, Jerôme Garin, Claude Cochet, Edmond Chambaz, and Jacques Baudier

From the Département de Biologie Moléculaire et Structurale du CEA,  DBMS-BRCE INSERM Unité 244, the  Laboratoire de Chimie des Proteines, DBMS-CP, CEN-G, 38054 Grenoble, the ^§ Laboratoire de Biophysique, UFR des Sciences Pharmaceutiques, 74 Route du Rhin, 67401 Illkirch, and the ^¶ Centre International de Recherche sur le Cancer, 69372 Lyon, France

The glial-derived calcium-binding protein S100B can be secreted to act as a neurotrophic factor or a mitogen, stimulating proliferation of glial cells. The extracellular S100B activities rely on the oxidation of the protein cysteine residues (Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7136-7139; Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989) J. Cell Biol. 109, 3063-3071). Here we show that oxidation of the S100B cysteine residues, Cys-68 and Cys-84, induces a conformational change in the protein structure, unmasking a canonical CKII phosphorylation site located within the typical EF-hand calcium-binding site II. Intrasubunit disulfide-bridged S100B monomer and disulfide-bonded S100B dimer are phosphorylated by the catalytic CKII- subunit on Ser-62 with a K_m of 0.5 µM and a V_max of 10 pmol/min/100 pmol of S100B. Oxidized S100B is the best in vitro CKII- substrate identified so far. Next we show that intrasubunit disulfide-bridged S100B monomer is the most potent S100B species to stimulate [³H]thymidine uptake by C6 glial cells in culture. In addition, the phosphorylated intrasubunit disulfide-bridged S100B monomer retains apparent mitogenic activity toward C6 glial cells, and hence, ³²P-labeled S100B should be a useful probe for characterizing the mechanisms by which extracellular oxidized S100B functions. Finally, we show that formation of intrasubunit disulfide-bridged S100B monomer is stimulated by peroxynitrite anion, suggesting that production of mitogenic S100B species could be enhanced in neuropathology associated with peroxynitrite anion production.