S100b(ββ) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner

Abstract

S100B(ββ) is a dimeric Ca2+ -binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19- 31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ, 1992, Proc Natl Acad Sci USA 89:11627-11631), S100B(ββ) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 ± 7 μM) in a Ca2+ - dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(ββ) in the presence of Ca2+ (IC50 = 17 ± 5 μM; IC50 = 1 ± 0.5 μM, respectively). At a minimum, the C-terminal EF-hand Ca2+ -binding domain (residues 61-72) of each S100β subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ((Ca)IC50 = 29.3 ± 17.6 μM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+ -binding domain of S100B (ββ).