Prorein Science zyxwvutsrqponm (1998), 7794-798. Cambridge University Press. Printed in the USA. Copyright zyxwvutsrqpon 0 1998 The Protein Society zyxwvutsrqp FOR zyxwvutsr THE RECORD S 1OOB (pp) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner PAUL T. WILDER, RICHARD R. RUSTANDI, ALEXANDER C. DROHAT, AND DAVID J. WEBER Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Mayrland 21201 (RECEIVED September 23, 1997; ACCEPTED November 13, 1997) Abstract: S100B(PP) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphor- ylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation do- mains 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 Nut1 zyxwvutsrqp Acud Sci USA 89:11627-11631), S100B(PP) binds the p53 peptide and inhibits its PKC-dependent phosphory- lation (IC50 zyxwvutsrqpo = 10 f 7 p M ) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regu- latory domain peptide were inhibited by SlOOB(PP) in the pres- ence of Ca2+ (IC50 = 17 zyxwvutsr f 5 pM; IC50 = 1 + 0.5 pM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (res- idues 61-72) of each Sloop subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition = 29.3 * 17.6 pM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+- binding domain of SlOOB(PP). Keywords: calcium-binding protein; p53; phosphorylation; PKM; protein kinase C; SIOOB; Sl00 protein Reprint requests to: David J. Weber, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 108 N. Greene St., Baltimore, Maryland 21201; e-mail, weber@noe.ab.umd.edu. Abbreviations: PKC, protein kinase C; PKM, catalytic subunit of PKC; [Ser-25]PKC( 19-31) peptide, peptide derived from the pseudo-substrate regulatory domain of PKCa (residues 19-31) with a serine at position 25 replacing the wild-type alanine (House & Kemp, 1987); PSCBD, phos- phorylation site/calmodulin binding domain; TCA, trichloroacetic acid: GFAP, glial fibrillary acidic protein; MARCKS, myristoylated alanine-rich C kinase substrate; MRF', MARCKS-related proteins; Nm, neuromodulin; DTT, dithiothreitol; PME, P-mercaptoethanol; IC, inhibition constant; BCA, bicinchoninic acid; p53, tumor suppressor protein; SMP, SlOO protein- modulated phosphoprotein; Sloop, a subunit of dimeric SlOOB(PP); SlOOB(PP), dimeric SlOOB with noncovalent interaction at the dimer interface. The SlOO protein family is a highly conserved group of Ca2+- binding proteins, and the principal member, SlOOB, was first dis- covered as a major constituent of glia (Moore, 1965). However, SlOOB is now known to be expressed in several tissues and cell lines including malignant tumors (Donato, 1991; Suzushima et al., 1994; Takashi et al., 1994; Zimmer et al., 1995). Although the precise mechanisms for intra- and extra-cellular functions of S 1 OOB are not well understood, processes such as neurite extension, Ca2+ flux, cell growth, apoptosis, energy metabolism, and protein phos- phorylation are all thought to be modulated in some manner by SlOOB (for reviews, see Kligman & Hilt, 1988; Donato, 1991; Zimmer et al., 1995; Schafer & Heizmann, 1996). The reduced homodimeric protein, S 100B(PP), is one of the best characterized proteins of the SlOO family. The solution struc- ture of reduced apo-SlOOB(PP) shows that two subunits associate tightly (KD < 500 pM) (Drohat et al., 1997) through extensive hydrophobic interactions to form a compact dimer with a highly charged surface (Amburgey et al., 1995; Drohat et al., 1996; Kilby et al., 1996). The general model for S 100-target protein inter- actions is similar to that of other CaZf-binding proteins, such as calmodulin and troponin C. Asforthese proteins, S100B(PP) undergoes a conformational change upon binding Ca2+ that pro- motes its interaction with a variety of target proteins (Kligman & Hilt, 1988; Drohat et al., 1996; Chaudhuri et al., 1997). For ex- ample, the Ca2+-dependent binding of SlOOB(PP) to micro- tubules (Bianchi et al., 1993), GFAP (Bianchi et al., 1994), and p53 (Baudier et al., 1992) prevents oligomerization for each of these proteins. SlOOB(PP) is also known to inhibit the PKC- dependent phosphorylation of 7-protein (Baudier et al., 1987), neuromodulin (Lin et al., 1994; Sheu et al., 1994), MARCKS (Albert et al., 1984), SMP (S100 Protein-Modulated Phosphopro- tein) (Pate1 et al., 1983), and p53 (Baudier et al., 1992; Wilder & Weber, 1996) by binding to the phosphorylation site of these PKC substrates. To further characterize the interaction of S100B(PP) with target proteins, we examined the interaction of SlOOB(PP) with peptides derived from the tumor suppressor protein, p53, neuromodulin, and the regulatory subunit of PKC. Previously, Baudier and co- workers showed that full-length p53 is a substrate for PKC in vivo 794