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Inhibition of p53 Transcriptional Activity by the S100B Calcium-binding Protein*

Open AccessPublished:September 14, 2001DOI:https://doi.org/10.1074/jbc.M104379200
      The levels of S100 Ca2+-binding proteins correlate with the progression of certain tumors, but their role, if any, in carcinogenesis is still poorly understood. S100B protein associates with both the p53 oligomerization domain (residues 325–355) and the extreme C terminus of the tumor suppressor p53 (residues 367–392). Consequently, S100B inhibits p53 tetramer formation and p53 phosphorylation mediated by protein kinase C, on p53 C-terminal end. In this report, we show that the S100B protein decreases p53 DNA binding and transcriptional activity. The effect of S100B is reflected in vivo by a reduced accumulation of p53, p21, and MDM2 protein levels in co-transfection assays and in response to bleomycin. The S100B can still interact with p53 in the absence of p53 extreme C-terminal end and reduce the expression of p53 downstream effector genes. These data indicate that S100B does not require p53 extreme C-terminal end to inhibit p53 activity. Collectively, these findings imply that elevated levels of S100B in tumors such as astrocytomas and gliomas could inhibit p53 functions and contribute to cancer progression.
      PKC
      protein kinase C
      CAT
      chloramphenicol acetyltransferase
      DTT
      dithiothreitol
      The S100 proteins are dimeric Ca2+-binding proteins (∼10 kDa/subunit) initially characterized by their solubility in 100% ammonium sulfate (S100) (
      • Moore B.W.
      ). Deregulation of Ca2+ homeostasis has been associated with different pathologies including neurodegenerative disorders, hypertension, and cancer (
      • Schafer B.W.
      • Heizmann C.W.
      ). The S100 proteins are overexpressed in many tumor cells and have been used as a marker for the classification of tumors (
      • Pedrocchi M.
      • Schafer B.W.
      • Mueller H.
      • Eppenberger U.
      • Heizmann C.W.
      ). A possible role for the S100 proteins in carcinogenesis has often been suspected, but their specific involvement is still ill defined. Evidence has indicated that S100B interacts with the tumor suppressor p53 (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ). p53 plays a pivotal role in the maintenance and regulation of normal cellular functions, and its inactivation can affect cell cycle checkpoints, apoptosis, gene amplification, centrosome duplication, and ploidy (
      • Ko L.J.
      • Prives C.
      ). p53 interacts with a number of proteins to mediate its pleiotropic effects. The interactions of p53 with S100 calcium-binding proteins are of particular interest because like p53, the S100 proteins affect cell cycle progression, are overexpressed in numerous tumor cells, and are associated with tumor progression (
      • Schafer B.W.
      • Heizmann C.W.
      ). The S100B protein interacts with the p53 C-terminal end and inhibits both,p53 tetramerization and phosphorylation by PKC1 (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ). Because these two events are known to be important for p53 activation (
      • Giaccia A.J.
      • Kastan M.B.
      ), we wanted to determine the effect of S100B on p53 transcriptional activity in vivo. Our data indicate that overexpression of S100B can reduce p53 transcriptional activity by more than 50%. This effect is correlated with a decrease in p53 DNA binding activity and a reduction in the accumulation of MDM2 and p21 protein levels. Interaction of the S100B protein with p53 may thus impede p53 cellular functions. Such an interaction could especially be detrimental in astrocytomas and gliomas, where S100B levels are significantly elevated (
      • Castets F.
      • Griffin W.S.
      • Marks A.
      • Van Eldik L.J.
      ).

      DISCUSSION

      In this report, we present evidence that S100B can reduce p53 DNA binding and transcriptional activity. S100B interacts with the p53 C-terminal end and inhibits both p53 tetramerization and phosphorylation in vitro (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ,
      • Wilder P.T.
      • Rustandi R.R.
      • Drohat A.C.
      • Weber D.J.
      ,
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ). These two events are known to be important for p53 transcriptional activation (
      • Giaccia A.J.
      • Kastan M.B.
      ) and suggest that in vivo S100B could interfere with p53 functions. Our data (Fig. 1) indicate that recombinant S100B protein reduces p53 DNA binding activity stimulated by an antibody (pAb421) directed against the residues 371–380 of the p53 C-terminal end. These data suggested that S100B competes with the p53 antibody pAb421 binding site. This possibility is supported by a recent NMR study (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ) showing a direct interaction between S100B and a p53 peptide (residues 367–388) derived from the p53 C-terminal end, which overlaps with the pAb421 epitope (residues 371–380). However, in contrast to pAb421, S100B does not stimulate p53 DNA binding activity (Fig. 2). This would suggest that the S100B interaction with the p53 C-terminal end is different from the interaction with the pAb421. In fact, S100B forms dimers (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ) and also interacts with the p53 oligomerization domain (
      • Delphin C.
      • Ronjat M.
      • Deloulme J.C.
      • Garin G.
      • Debussche L.
      • Higashimoto Y.
      • Sakaguchi K.
      • Baudier J.
      ). The occupation of both sites, oligomerization and C-terminal end, by S100B could thus lead to disruption of the p53 tetramer formation and reduction of p53 DNA binding activity (Figs. 1 and 2). A transition of the p53 latent form to the active DNA binding form mediated by pAb421 has been shown to occur for p53 tetramers (
      • Waterman J.L.
      • Shenk J.L.
      • Halazonetis T.D.
      ). By inhibiting p53 tetramer formation and occupying the C-terminal end, S100B may interfere with the p53 activation and reduce DNA binding activity (Fig.1). This hypothesis is supported by the more than 50% reduction of p53 transcriptional activity measured in the presence of S100B (Fig. 3). However, the reduction of p53 DNA binding activity observed in the absence of p53 extreme C-terminal end (Fig. 2) suggests that interaction with the p53 C-terminal end alone is not sufficient to mediate the S100B inhibitory effect.
      Our data indicate that the p53 extreme C-terminal end is not required for interaction with S100B (Fig. 5) or reduction of p53 downstream effector genes accumulation (Fig. 4 A). It is possible thatin vivo, modifications of p53 C-terminal end hinders S100B binding. The extreme p53 C-terminal end (residues 367–392) contains several phosphorylation and acetylation sites important for p53 functions. A recent analysis by NMR (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ) has indicated that S100B sterically blocks two important PKC phosphorylation sites (Ser-376 and Thr-377). This study (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ), performed on non-phosphorylated peptide, could either explain the inhibitory effect of S100B on p53 phosphorylation by PKC (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ,
      • Wilder P.T.
      • Rustandi R.R.
      • Drohat A.C.
      • Weber D.J.
      ) or indicate that phosphorylation of these sites may change the interaction. A recent report (
      • Youmell M.
      • Park S.J.
      • Basu S.
      • Price B.D.
      ) has indicated that p53 mutation at these PKC phosphorylation sites (S376A and T377A) reduces p53 transcriptional activity by ∼38 and 51% respectively. This is very similar to what we observed here when we transfected p53 with S100B (Fig. 3). Moreover, mutation at Ser-378, another PKC site on p53, did not affect p53 transcriptional activity (
      • Youmell M.
      • Park S.J.
      • Basu S.
      • Price B.D.
      ). This Ser is well exposed and not blocked by S100B (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ). The level of p53 C-terminal end phosphorylation could thus influence the interaction with S100B and explain the partial inhibitory effect encountered in vivo. Alternatively, interaction of other regulatory proteins such as XP-B, XP-D, or Ref-1 (
      • Jayaraman L.
      • Murthy K.G.
      • Zhu C.
      • Curran T.
      • Xanthoudakis S.
      • Prives C.
      ) with p53 C-terminal end may reduce S100B interaction with p53 and explain why only a partial inhibitory effect is observed.
      As mentioned earlier, S100B inhibits p53 phosphorylation by PKC and formation of p53 oligomers. These two activities may actually be link because phosphorylation of p53 by another kinase, hCHK1, requires that p53 be oligomeric (
      • Shieh S.Y.
      • Ahn J.
      • Tamai K.
      • Taya Y.
      • Prives C.
      ). Moreover, co-transfection of p53 with antisense hCHK1 reduces p53 levels (
      • Shieh S.Y.
      • Ahn J.
      • Tamai K.
      • Taya Y.
      • Prives C.
      ). This is reminiscent of what we are observing with S100B (Fig. 4). By preventing oligomerization of p53, S100B could reduce phosphorylation and stabilization of p53 by not only PKC but also by other kinases such as hCHK1 (
      • Shieh S.Y.
      • Ahn J.
      • Tamai K.
      • Taya Y.
      • Prives C.
      ). This possibility seems very likely because S100B also reduces the accumulation of p53 Δ30, which lacks the PKC phosphorylation site (Fig.4 A).
      Regardless of the mechanism(s) involved, our findings indicate that S100B prevents p53 from regulating its downstream effector genes (Fig.4) and could consequently alter p53 cellular functions. Our data seem to oppose a recent report (
      • Scotto C.
      • Deloulme J.C.
      • Rousseau D.
      • Chambaz E.
      • Baudier J.
      ) describing the cooperation of S100B with p53-mediated cellular functions. The study presented by Baudier's team (
      • Scotto C.
      • Deloulme J.C.
      • Rousseau D.
      • Chambaz E.
      • Baudier J.
      ) used a temperature sensitive mutant (p53Val135) transfected in rodent cells. In such a system, a mixture of p53 wild type and mutant conformation exists. Since the p53 mutation is localized at amino acid 135 and that S100B interacts with p53 at the C-terminal end between amino acid residues 319–393, it seems probable that S100B can not discriminate between the two p53 forms. The effects observed by this group could simply be due to the release of wild type p53 molecules from p53 wild type-mutant heterodimers and not be a direct effect of S100B on p53 activity. The cell line used here, H1299, has a null genotype for p53 (
      • Kastan M.B.
      • Zhan Q.
      • el-Deiry W.S.
      • Carrier F.
      • Jacks T.
      • Walsh W.V.
      • Plunkett B.S.
      • Vogelstein B.
      • Fornace Jr., A.J.
      ), therefore the only p53 present in our system was the wild type form (full-length or deletions) that we transfected. Moreover, as mentioned above, the interaction of S100B with p53 has been shown to disrupt p53 oligomerization and phosphorylation (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ). Because those two events are known to be important for p53 activation (
      • Giaccia A.J.
      • Kastan M.B.
      ), one may anticipate that interaction with the S100B protein would interfere with p53 cellular functions. The data presented here and the observations mentioned above (
      • Rustandi R.R.
      • Baldisseri D.M.
      • Weber D.J.
      ,
      • Youmell M.
      • Park S.J.
      • Basu S.
      • Price B.D.
      ) support the idea that S100B inhibits p53 activity. Moreover, a recent report (
      • Grigorian M.
      • Andresen S.
      • Tulchinsky E.
      • Kriajevska M.
      • Carlberg C.
      • Kruse C.
      • Cohn M.
      • Ambartsumian N.
      • Christensen A.
      • Selivanova G.
      • Lukanidin E.
      ) has indicated that S100A4, another member of the S100 protein family decreased p53 DNA binding and transcriptional activity.
      In summary, our data indicate that S100B inhibits p53 functions. The p53 extreme C-terminal end is not required for this effect. Since S100B has been shown to interact with p53 oligomerization domain and prevent the formation of oligomers (
      • Baudier J.
      • Delphin C.
      • Grunwald D.
      • Khochbin S.
      • Lawrence J.J.
      ), this mechanism is a likely explanation for the inhibitory effect observed here. However, we can not rule out the possibility that other p53 domains are involved. S100B is a very abundant protein expressed at high levels in certain tumors (
      • Castets F.
      • Griffin W.S.
      • Marks A.
      • Van Eldik L.J.
      ), its inhibitory effect on the low abundance p53 tumor suppressor protein may contribute to cancer progression.

      Acknowledgments

      We thank Dr. Steven Hirschfeld for a careful reading of this manuscript and insightful discussion.

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