Characterization of the Tumor Suppressor Protein P53 As a Protein Kinase C Substrate and a Sloob-Binding Protein

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 11627-11631, December 1992 Biochemistry Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a SlOOb-binding protein JACQUES BAUDIER*, CHRISTIAN DELPHIN, DIDIER GRUNWALD, SAADI KHOCHBIN, AND JEAN JACQUES LAWRENCE Laboratoire de Biologie Moleculaire du Cycle Celiulaire, Institut National de la Sante et de la Recherche M6dicale Unite 309, CEN-G-85X, 38041 Grenoble Cedex, France Communicated by Daniel E. Koshland, Jr., August 17, 1992

ABSTRACT We report here that the negative cell cycle explained through the binding ofthe calcium binding proteins regulator protein p53 is an in vivo and in vitro substrate for to the PKC phosphorylation site domain (13). Because of the protein kinase C, a cellular receptor for the tumor-promoter similarities between the basic C-terminal domain of p53 and phorbol esters. We also demonstrate that p53 interacts in a the PKC phosphorylation site domain on MARCKS protein, calcium-dependent manner with SlOOb, a member of the S100 we investigated the possibility that p53 might also be a PKC protein family involved in cell cycle progression and cell substrate that interacts with SlOOb and CaM. differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and SlOOb was utilized for the purification of cellular and EXPERIMENTAL PROCEDURES recombinant murine p53 by affinity chromatography with Cell Lines and Culture. Murine erythroleukemia cells SlOOb-Sepharose. Furthermore, and of particular interest, we (MELCs), clone F4NWO, were maintained in culture in have shown that purified p53 undergoes temperature- minimum essential medium (MEM) supplemented with 10%o dependent oligomerization and that the interaction between (vol/vol) fetal calf serum and 1 mM 5% (wt/vol) glutamine. SlOOb and p53 not only induces total inhibition of p53 oligo- cDNA cloning of the p53 mRNA in our MELC strain has merization but also promotes disassembly ofthe p53 oligomers. revealed no mutation in the five conserved domains of the We suggest that these effects result from the binding of SlOOb protein where the transforming mutations generally occur (1), to the multifunctional basic C-terminal domain of p53 and but a 9-amino acid deletion (residues 255-262) was found propose that p53 may be a cellular target for the S100 protein when compared to the wild-type protein (unpublished data). family members involved in the control of the cell cycle at the Protein Purification and Antibodies. Rat-brain PKC iso- Go-G1/S boundary. forms were purified as described (14). Bovine brain SlOOb protein was purified as described (15). SlOOb and CaM- The cell cycle is composed of a series of steps that can be Sepharose columns were prepared as in ref. 16. Monoclonal negatively or positively regulated by various factors. Chief antibodies PAB-240, PAB-248, PAB-242, and PAB-421 are among the negative regulators is the p53 protein (1). The p53 from hybridoma cell supernatants (17, 18). gene was initially proposed to be an oncogene, but more Metabolic Labeling, Immunoprecipitation, and Autoradiog- recent work has shown that only mutant forms of p53 elicit raphy. Approximately 3 X 107 exponentially growing MELCs neoplastic transformation in vitro and that wild-type p53, on were preincubated in 50 ml of phosphate-free MEM with 5% the contrary, could inhibit oncogene-mediated focus forma- glutamine for 90 min. The cells were pelleted by centrifugation, thus acting as a tumor suppressor gene (2). Several tion and resuspended in 5 ml of phosphate-free MEM plus properties ofp53 have been described (for review, see ref. 3). [32P]orthophosphate (1 mCi/ml; 1 Ci = 37 GBq). Cells were p53 is a cytosolic phosphoprotein that has to translocate to labeled for 90 min, split 1:2, and treated with 2 1.l of dimethyl the nucleus for cellular activity (4). p53 can be phosphory- sulfoxide or with 2 p.l of a phorbol 12-myristate 13-acetate lated by cdc2 kinase (5) and casein kinase 11 (6). p53 can also (PMA) solution (3 mM) in dimethyl sulfoxide. After 10 min, form large oligomers and p53 oligomerization has been di- cells were washed twice with phosphate-buffered saline rectly linked to the cellular function of the protein (7). (PBS) and lysed in 1 ml of lysis buffer [40 mM Tris-HCl, pH Analysis ofthe p53 primary structure reveals that the protein 7.5/100 mM NaCl/1 mM NaF/aprotinin (10 pug/ml)/0.5% has an extended basic C-terminal region that is predicted to Triton X-100]. After sonication and centrifugation to remove form an amphipathic helix-like structure and is probably insoluble material, p53 was immunoprecipitated with a mix- responsible for its binding to DNA and the formation ofstable ture ofmonoclonal antibodies PAB-242, PAB-240, and PAB- p53 oligomers (8, 9). This putative DNA-binding and oligo- 248. The immunoprecipitate was analyzed by SDS/PAGE on merization-promoting domain on p53 shows a striking simi- an 8% gel and autoradiography. larity with predicted protein kinase C (PKC) phosphorylation In Vitro PKC Phosphorylation Assays. Standard phosphor- sites (10). In addition, this domain resembles in some respects ylation assays were performed in 40 mM Tris HCl (pH 7.5) at the PKC phosphorylation and calmodulin (CaM)-binding 350C. The reaction mixture (100 pl) contained 1 puM p53, 80 domain on the myristoylated alanine-rich C kinase substrate p.M [32P]ATP, and 3 mM MgCl2. CaCl2 (0.2 mM), phospha- (MARCKS) protein (11) (see also Fig. 7). MARCKS is a tidylserine (2 pug), and diolein (0.25 ,ug) were included in the major PKC substrate rapidly phosphorylated in a wide vari- assay or omitted as indicated. The reaction was stopped by ety ofcell types in response to growth factor stimulation (11). adding SDS stop solution [5% (wt/vol) SDS/25% (vol/vol) In vitro MARCKS phosphorylation is strongly inhibited by glycerol/500 mM Tris-HCI, pH 6.8]. The proteins were calcium binding proteins of the "EF-hand" type, S100b, and separated by SDS/PAGE on an 8% gel and the gels were to a lesser extent by CaM (12). This inhibition has been Abbreviations: MELC, murine erythroleukemia cell; PKC, protein The publication costs ofthis article were defrayed in part by page charge kinase C; MARCKS, myristoylated alanine-rich C kinase substrate; payment. This article must therefore be hereby marked "advertisement" CaM, calmodulin; PMA, phorbol 12-myristate 13-acetate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11627 Downloaded by guest on September 28, 2021 11628 Biochemistry: Baudier et al. Proc. Natl. Acad. Sci. USA 89 (1992)

fixed and processed for autoradiography. 32p incorporation Temperature-Dependent Olimerization of p53. p53 (0.7 into p53 was quantified by liquid scintillation counting ofthe mg/ml) in 40 mM Tris-HCI, pH 7.6/0.16 mM CaCl2 was protein excised from the gel. maintained in an ice-water bath and then transferred to a The Binding of Cellular p53 to SlOb-Sepharose. Approxi- thermostated cell at 300C that contained an equal volume of mately 2.7 x 108 MELCs were lysed at 4TC in 8 ml of 50 mM buffer or different concentrations of S100b dialyzed against TrisHCl, pH 7.5/2 mM EGTA/200 mM NaCl/50 mM 40 mM Tris-HCl (pH 7.6). The increase ofA350 was recorded NaF/1% Triton X-100/aprotinin (60 ,ug/ml)/1 mM phenyl- in a trace mode as a function of time. methyl sulfonyl fluoride and sonicated. After centrifugation at 15,000 x g for 15 min, the supernatant was collected, RESULTS diluted 1:10 with buffer A (50 mM Tris-HCl, pH 7.5/4 mM dithiothreitol), and loaded onto a DE52 column (2 x 10 cm) Caaerizat of p53 as a PKC Substrate. The tumor equilibrated with buffer A. After washing the column with promoter phorbol ester PMA is an in situ activator of 100 ml ofbuffer A, the bound proteins were eluted with buffer intracellular PKC. To assess the role of PKC in phosphory- A plus 0.4 M NaCl. The eluted protein fractions were pooled lating p53, we compared the effect of PMA (0.1 MM) stimu- (50 ml), diluted with an equal volume ofbuffer A, adjusted to lation of Friend erythroleukemia cells on 32p labeling of p53 1 mM CaCl2, and loaded onto an Sl0Gb-Sepharose column (1 with that of control cultures. A constant increase in 32p x 8 cm) equilibrated with buffer A containing 200 mM NaCI incorporation could be observed in immunoprecipitated p53 and 1 mM CaCI2. The flow-through fractions were recycled from PMA-treated cells compared to control (Fig. 1A). The once on the column and the column was washed with 10 increase in 32p incorporation varied between 1.5- and 2-fold, column volumes of equilibration buffer. The bound proteins by densitometry scanning of the autoradiograms. were subsequently eluted with buffer A containing 200 mM In vitro phosphorylation studies of p53 using purified rat NaCl and 2 mM EGTA; 1-ml fractions were collected. brain PKC confirmed p53 as PKC substrate (Fig. 1B). Among Purification of Recombinant Murine p53. Approximately the different rat brain PKC isoforms (types I-III), the type II 3.8 x 108 Sf9 cells were infected with recombinant baculo- and type III phosphorylated p53 significantly, whereas type virus vEV55p53 (19). Forty-eight hours after infection, the I PKC was much less efficient (Fig. 1B). Although the cells were harvested, washed once with PBS, lysed in 100 ml amounts ofPKC isoforms used in the experiments presented in were not as in of deionized water 2 mM EGTA Fig. 1B equivalent, revealed by differences purified containing (pH 7.6) their autophosphorylation signal (Fig. 1B), a 1:2 to 1:5 and 4 mM dithiothreitol, sonicated, and immediately frozen dilution of the type H PKC preparation had no significant at -20°C. After thawing, the cell extract was maintained at effect on the extent of p53 phosphorylation, suggesting that 4°C, 1 M Tris-HCI (pH 7.6) was added to 40 mM (final differences in p53 phosphorylation by the various PKC concentration), and the extract was briefly sonicated and isoforms might reflect differences in specificity for p53. centrifuged at 15,000 x g for 15 min. The supernatant fraction In the absence of added kinase, we observed that the was loaded on a Q-Sepharose fast flow column (2 x 10 cm) purified recombinant p53 incorporated 32P when incubated equilibrated with buffer B (40 mM Tris HCI, pH 7.6/5 mM with [32P]ATP to a maximum stoichiometry of 0.05 mol of dithiothreitol). The column was serially washed with buffer B phosphate per mol of protein (Figs. 1B and 2A). This p53 plus 0.1 M NaCl, 0.2 M NaCI, and 0.4 M NaCl. Immunoblot phosphorylation could not be stimulated by Ca2+ and phos- analysis revealed that p53 was eluted with buffer B containing pholipids and, therefore, did not result from contamination of 0.4 M NaCI. The p53-containingfractions were pooled (40ml) and diluted 1:2 with buffer B. Halfofthe protein solution (40 A ml) was adjusted to 1 mM CaCl2 and loaded on a S100b- Sepharose column (1.5 x 10 cm) previously equilibrated with B buffer B containing 0.15 M NaCl and 1 mM CaC42. The column was washed with equilibrating buffer, and p53 protein was eluted with buffer B containing 2 mM EGTA and 0.15 M NaCl. The final purification step consisted of FPLC chro- - P K( matography on a Mono Q column using a linear gradient from 0 to 0.7 M NaCl. p53 was eluted from the Mono Q column as .- p3-i a symmetrical protein peak at 0.3 M NaCl. The protein was dialyzed against 40 mM Tris-HCl (pH 7.5) and stored at -200C until used. p53 protein concentrations were determined using the Bio-Rad protein assay system and bovine serum albumin as standard. Overlay Procedures and Immunodetection of Covalently Cross-Linked p53-SlOOb Complexes. Triplicate samples of purified human p53 (0.5 ug) were electrophoresed on 8% SDS/PAGE gel and electrotransferred to an Immobilon membrane. After blocking the membrane with 3% (wt/vol) bovine serum albumin in buffer C (20 mM Tris-HCl, pH 7.5/150 mM NaCl), the membrane was cut in three pieces and incubated 15 min with either buffer C or SlOOb (2 AM) in buffer C in the presence of 1 mM Ca2+ or in the presence of 1 mM EGTA. After washing the membranes with corresponding buffers to remove unbound S10Gb, the p53-SlOOb FIG. 1. Phosphorylation of p53 by PKC. (A) Immunoprecipita- complexes were then cross-linked with 1-ethyl-3-(3- tion of phosphorylated p53 from 32P-labeled MELCs. Lanes: 1, dimethylaminopropyl) carbodiimide hydrochloride (Pierce; 2 control cells; 2, cells treated with PMA. (B) In vitro phosphorylation of purified p53 by the rat-brain PKC isoforms. Purified p53 was mM in buffer C). The p53-SlOOb complexes were then incubated for 30 min in standard PKC assay conditions in the absence visualized by immunodetection using a rabbit anti-SlOOb (lane 1) or in the presence oftype I, type II, or type III PKC isoforms serum produced in our laboratory (at a 1:50 dilution) and (lanes 2-4, respectively). Positions ofthe phosphorylated p53 and of phosphatase-conjugated goat anti-rabbit IgG. the autophosphorylated PKC are indicated. Downloaded by guest on September 28, 2021 Biochemistry: Baudier et al. Proc. Nati. Acad. Sci. USA 89 (1992) 11629

A A B

Ca2+ . + + + --PKC PL PKC + + -+ _ ^ ~~~p53 P>, -4 S100b CaM 1 2 3) FiG. 2. In vitro phosphorylation of p53 by type II PKC: The effects of S100b. (A) Recombinant p53 was incubated S min in standard phosphorylation assay conditions in the absence (-) or f presence (+) of type II PKC and PKC cofactors, Ca2+ and phospholipid (PL), as indicated. Comparison of the effect of SlOOb and CaM on p53 phosphorylation in the absence of added kinase is also shown. (B) Recombinant p53 (1 was incubated 5 min in standard 1AM) I- o t Purification of the recombinant mu- assay Ca2+ and PKC phosphorylation conditions (plus phospholi- rine p t using S affinity chromatography. with type II PKC in the absence (lane 1) and in the presence pase) Purification of recombinant p5 3 involved Sil()ob- or of S100b at 0.9 AM (lane 2) 1.8 ,uM (lane 3). Sephtarose aind Mono Q chromatography columns. (A and B) The Sl100-Sepharose fractions the p53 with PKC. Whether this phosphorylation of p53 were ainalkzed by silver staining and immuno- results from an intrinsic autophosphorylation process or is blot. respectivel. Lanes: 1. sample loaded on due to a contaminating kinase that copurifies with p53 the S1iQh-Sepharose column: 2. flov-through remains to be determined. When an equimolar concentration fraction 3. protein fraction eluted with EGTA of S100b or CaM was added to p53 plus 0.1 mM Ca2+ prior and I5 miM NaGC: 4. protein traction eluted with \and SOO mM NaCl. Between A Lind to incubation with [32P]ATP, a total inhibition of the p53 EGTA B arc indicaited positions of the molecular masss phosphorylation occurred only in the presence of SlOOb; stand rds in kIla. C Amido black staining of CaM had no effect (Fig. 2A). the purified p53 4rig) elUted from the Mono Q As expected phosphorylation of p53 by type II PKC was column and analLvze d Kh SD) S PAGE. on a-i exclusively Ca2+- and phospholipid-dependent (Fig. 2A). In our assay conditions, the cofactor-dependent phosphorylation ofp53 was rapid at 35°C and was maximal by 10 min with abundance and revealed that several other proteins also an apparent stoichiometry of 0.65 mol of phosphate incor- bound to the SlOOb column (Fig. 3A); most of these proteins porated per mol of protein, suggesting a single phosphory- have been identified as PKC substrates (data not shown). lation site on the protein. For comparison, we also tested the binding of cellular p53 When SlOOb was present in the phosphorylation assay, a to CaM by affinity chromatography. Although significant 70% inhibition of the Ca2+/phospholipid-dependent PKC calcium-dependent binding of p53 on the CaM-Sepharose column was observed, most of the p53 was recovered in the phosphorylation ofp53 was observed for an S100b/p53 molar flow-through fractions, suggesting higher affinity of p53 for ratio of 1.8 (Fig. 2B). Because S100b has no effect on PKC the Ca2+-SlOOb than for Ca2+-CaM (data not shown). autophosphorylation (Fig. 2B) and does not inhibit phos- The SlOOb affinity chromatography method was then used phorylation ofother substrates [e.g., histone (16)], it is unlikely to purify murine p53 expressed in SF9 insect cells infected that this inhibition is due to an effect of S100b on the kinase with recombinant baculovirus vEV55pS3 (19). Recombinant itself but rather is a direct consequence of the interaction of murine p53 bound to the S10Db column in the presence of S10Db with p53. Unlike SlOOb, CaM had no significant effect Ca2+ and could be eluted with EGTA-containing buffer (Fig. on p53 phosphorylation by PKC (data not shown). 4). A significant amount of p53 (r5%) remained bound to the The Binding op53 to SlOOb. To confirm the binding ofp53 SlOOb-Sepharose in the presence ofEGTA and 150 mM NaCl to SlOOb protein, an affinity chromatography procedure was but was eluted with a higher NaCI concentration (500 mM), developed using MELC extract and SlOOb Sepharose (Fig. suggesting that it can interact with SlOOb in the absence of 3). All the p53 in the MELC extract was retained on the SlOOb Ca2+. Subsequent purification by anion-exchange chroma- column in the presence of Ca2+ and subsequently eluted tography on a Mono Q column resulted in purified p53 protein when EGTA replaced Ca2+ (Fig. 3B). Silver staining of the judged to be >95% pure when analyzed on an SDS/ p53-containing fractions did not detect p53 due to its low polyacrylamide gel and amido black staining (Fig. 4C). /A B

C.,N FIG. 3. Cellular p53 from MELCs bind to an S100b- Sepharose column. Analysis of fractions from the -4 S100b-Sepharose column chromatography by SDS/ PAGE on an 8% gel (5 gg of protein per lane) followed by either silver staining (A) or immunoblot analysis c , >a _ _m_ with PAB-421 monoclonal antibody (1:200 dilution) (B). (A) Lanes: 1, sample loaded on the S100b- U- Sepharose column; 2, flow-through fraction; 3, fraction eluted with EGTA. (B) Lanes: 1, sample loaded on the S100b-Sepharose column; 2, first flow-through fraction; 3, second flow-through fiaction; 4-9, fractions eluted with EGTA. The silver-stained band on the left side of A and the arrows in B correspond to the molecular mass standards 30, 46, 69, and 92 kDa, respectively. Downloaded by guest on September 28, 2021 11630 Biochemistry: Baudier et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3 A ®FE)OF [A L® G FQ F -I NI E A G MARCKS 157 177 _ (j(.; S Y Li TIjK G Q S T S[B HEK T M Vill V Mouse p53 361 382 S H LI[ BSiIJ G Q S T S[ HE iK L M F[3 T E Human p53 367 388 _-, 67 FIG. 7. Comparison ofthe C-terminal basic amino acid sequence -45 of mouse and human p53 with that of the PKC and CaM-binding domain on MARCKS. Sequences have been aligned manually to compare positions of the putative serine/threonine PKC phosphorylation sites on p53 (boldface type) with that of the phosphorylated serine residues (circled) on MARCKS. Note the striking similarity in 31n. position of the basic amino acids that are essential for PKC phosphorylation but also presumably promote association with the acidic CaM or S1OOb molecules as demonstrated for other proteins (20, 21). ofpurified p53 in 40 mM Tris HCI (pH 7.6) showed abnormal light scattering relative to that of a solution at 4eC, FIG. 5. Human recombinant p53 binds directly to the SlOOb. (A) indicating Coomassie blue staining ofpurified human recombinant p53 (0.5jfg) temperature-dependent aggregation (Fig. 6A). In Fig. 6B the analyzed by SDS/PAGE on an 8% gel. Lanes: 1, purified p53; 2, time-dependent changes in light scattering at 350 nm are molecular mass standards. (B) The direct interaction between puri- presented for a p53 solution when the temperature was fied human p53 and S100b is demonstrated by an overlay procedure shifted from 40C to 30TC. In parallel experiments, we found and immunodetection ofthe covalently cross-linked p53-S100b com- that the p53 aggregation was independent of the ionic plexes. Lanes: 1, control lane corresponding to p53 alone; 2, p53 strength, although larger p53 oligomers were formed in the incubated with S100b in the presence of1 mM Ca2+; 3, p53 incubated presence of0.3 M NaCl than in its absence. ATP or GTP has with S100b in the presence of 1 mM EGTA. On the right, positions no effect on the oligomerization of p53. of molecular mass standards are indicated (kDa). In A and B, The p53 oligomers formed at 300C were sufficiently large to arrowheads indicate position of p53. be sedimented at 200,000 x g (Fig. 6C). same When increasing amounts of SlOb were added to p53, in The purification protocol was used to purify human the presence of 0.1 mM C42+, prior to shifting the tempera- recombinant p53 (Fig. 5A). The SlOOb-Sepharose column ture from 40C to 300C, the oligomerization of p53 was might be useful for purification of mammalian p53 in general. inhibited in a concentration-dependent manner (Fig. 6B) and The binding ofp53 to the SlOOb-Sepharose column through p53 could not be sedimented by centrifigation (Fig. 6C). a direct interaction with SlOOb was confirmed by an overlay Furthermore, when SlOOb plus Ca2+ was added to oligomer- procedure and covalent crosslinking of the p53-SlOOb com- ized p53 at 300C, light scattering of the p53 protein solution plexes (Fig. 5). As expected the p53-S10Gb complexes formed at 350 nm decreased within 2 min to reach the level recorded in the presence of Ca2+ but not in the presence of EGTA. at 40C (Fig. 6B). These results indicate that S10Gb can interact The Interaction Between p53 and S1Ob Regulate p53 with p53 to prevent its oligomerization and that it can also Olgomerization in Vitro. At 300( the UV absorption spectrum induce disassembly of p53 oligomers, perhaps by sequester-

p -~-, p

* U U m U U

0) .2 F.-... a) ! B T

n, 4- 053 at , < , 1. O .1 ,.--

I '....

e .. S1OOb .- 260 300 340 1 U6 10 14 Wavelength, n-r Time. mil FIG. 6. Temperature-dependent oligomerization ofp53 and the effects ofSlOOb. (A) UV absorption spectra ofp53(0.11 mg/ml) at4'C (curve 1) and 30"C (curve 2). (B) The temperature-dependent changes in A35oofthe p53 protein were studied in the absence (solid line) or in the presence of various concentrations of SlOOb (dashed lines) as indicated (IAM). Also shown in B is the effect of S10Gb ( I ) plus 0.2 mM CaCl2, or plus 0.1 mM EGTA on the changes in A35o of the oligomerized p53 solution at 30"C (SlOOb/p53 molar ratio, 4). (C) The effect of SlOOb on the sedimentation of the p53 oligomers. p53 (2 t.M) (lanes 1) was mixed with SlOOb (8 AtM) plus 0.2 mM CaCl2 (lanes 2) or 0.1 mM EGTA (lanes 3). After 11 min at 250C, the proteins were centrifuged at 25TC for 30 min at 200,000 x g in a Beckman TL 100 centrifuge and the proteins in the supernatant fractions (S) and in the pellets (P) were analyzed by SDS/PAGE on an 8% gel and amido black staining. Downloaded by guest on September 28, 2021 Biochemistry: Baudier et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11631 ing p53 monomers. Using this approach we also confirmed It would be worthwhile to study the possible association of that, in the absence of NaCl, the interactions between SlOOb SlOOb or ofthe other members ofthe S100 protein family with and p53 can occur in the absence ofCa2+ but are strengthened p53 in living cells and to investigate whether these interac- when Ca2+ is present. SlOOb induced partial disassembly of tions function in regulating p53 oligomerization in the cyto- p53 oligomers in the presence of0.1 mM EGTA, as indicated plasm and/or in controlling its nuclear transport (4). In this by a decrease in light scattering of the protein solution (Fig. respect it is significant to note that the putative S100-binding 6B) and a reduced sedimentation of p53 oligomers (Fig. 6C). domain on p53 overlaps with two nuclear localization signals that mediate the migration of p53 to the cell nucleus (24). DISCUSSION We acknowledge Mr. H. Gleyzolle (Institut National de la Sante et de la Recherche Mddicale U244) for his contribution to the The characterization of p53 as a PKC substrate that also purification of the PKC isoforms. We thank Dr. J. Gannon for binds to SlOOb lends support to our observation that the preliminary amino acid sequence ofthe p53 phosphopeptide, Drs. D. C-terminal basic domain of p53 is similar in certain respects Pigott, and R. D. Cole for comments on the manuscript, and Drs. J. to the PKC- and CaM/SlOOb-binding domain on the MARCK Jenkins and D. O'Reilly for providing us with p53 baculovirus. This protein (Fig. 7). work is supported by a grant (6777) from the "Association pour la Phosphorylation of p53 by PKC has been demonstrated in Recherche sur le Cancer" to J.B. situ and in vitro. The limited stimulation of p53 phosphory- 1. Levine, A. J., Momand, J. & Finlay, C. A. (1991) Nature (London) lation in MELCs by PMA suggests that in situ phosphory- 351, 453-456. lation of p53 will not only depend on the activation of the 2. Finlay, C. A., Hinds, P. W., Tan, T. H., Eliyahu, D., Oren, M. & enzyme but also on the accessibility ofp53 to the enzyme that Levine, A. J. (1988) Mol. Cell. Biol. 8, 531-539. 3. Lane, D. P. & Benchimol, S. (1990) Genes Dev. 4, 1-8. could be regulated by intracellular factors and/or by its 4. Gannon, J. V. & Lane, D. P. (1991) Nature (London) 349,802-806. appropriate cellular localization as observed for other PKC 5. Addison, C., Jenkins, J. R. & Sturzbecher, H. W. (1990) Oncogene substrates (22, 23). Although we have not yet been able to 5, 423-426. determine the PKC phosphorylation site on p53, preliminary 6. Meek, D. W., Simon, S., Kikkawa, V. & Eckhart, W. (1990) EMBO J. 5, 3253-3260. studies have shown that V8 proteolysis ofthe phosphorylated 7. Milner, J. & Medcalf, E. A. (1991) Cell 65, 765-774. p53 generated a single phosphopeptide whose sequence be- 8. Kern, S. E., Kinzler, K. W., Baker, S. J., Nigro, J. M., Rotter, V., gan at residue 350, thus including the predicted PKC phos- Levine, A. J., Fredman, P., Prives, C. & Vogelstein, B. (1991) phorylation site. Further work should be done on the bio- Oncogene 6, 131-136. 9. Milner, J., Medcalf, E. A. & Cook, A. C. (1991) Mol. Cell. Biol. 11, logical meaning ofthe PKC phosphorylation ofp53 in a region 8-15. of the protein that is suspected to sustain several functional 10. Kennelly, P. J. & Krebs, E. G. (1991) J. Biol. Chem. 266, 15555- properties such as oligomerization (9), nuclear transport (24), 15558. and DNA binding (8). 11. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P. & Black- The observed of cellular and to shear, P. J. (1989) Proc. Natl. Acad. Sci. USA 86, 4012-4016. binding recombinant p53 12. Albert, K. A., Wu, W. C.-S., Nairn, A. C. & Greengard, P. (1984) SlOOb-Sepharose in the presence of calcium, and to a lesser Proc. Natl. Acad. Sci. USA 81, 3622-3625. extent to CaM-Sepharose, further supports the idea that the 13. Graff, J. M., Young, T. M., Johnson, J. D. & Blackshear, P. (1989) basic C-terminal domain on p53 and the PKC domain on J. Biol. Chem. 264, 21818-21823. MARCKS are functionally related. Indeed, as was shown for 14. Pelosin, J. M., Vilgrain, I. & Chambaz, E. M. (1987) Biochem. Biophys. Res. Commun. 147, 382-391. MARCKS (13), it is probably this domain on p53 that also 15. Baudier, J., Holtzcherer, C. & Gerard, D. (1982) FEBS Lett. 148, serves in binding SlOOb. The SlOOb-mediated inhibition of 231-234. both p53 phosphorylation and oligomerization can, therefore, 16. Baudier, J., Mochley-Rosen, D., Newton, A., Lee, S. H., Kosh- be explained through the same basic process-i.e., the bind- land, D. E. & Cole, R. D. (1987) Biochemistry 26, 2886-2893. ing of SlOOb to the C-terminal basic amphiphilic amino acid 17. Harlow, E., Crawford, L. V., Pim, D. C. & Williamson, N. M. (1981) J. Virol. 39, 861-869. sequence on p53, blocking the accessibility of the phosphor- 18. Yewdell, J. W., Gannon, J. V. & Lane, D. P. (1986) J. Virol. 59, ylation site for the activated PKC, and preventing interac- 444-452. tions between p53 monomer (9). 19. O'Reilly, D. R. & Miller, L. K. (1988) J. Virol. 62, 3109-3119. The observed interaction between p53 and SlOOb might 20. Herring, B. P. (1991) J. Biol. Chem. 266, 11838-11841. also provide a clue for understanding, at the molecular level, 21. Bagchi, I. C., Huang, Q. & Means, A. R. (1992)J. Biol. Chem. 267, the of 3024-3029. relationship Ca2+, cell-cycle-associated calcium- 22. Rosen, A., Nairn, A. C., Greengard, P., Cohn, Z. A. & Aderem, A. binding proteins of the S100 family, and the G0-G1/S tran- (1989) J. Biol. Chem. 264, 9118-9121. sition (for reviews, see refs. 25 and 26). 23. Hourbe, D., Duportail, G., Deloulme, J. C. & Baudier, J. (1991) J. SlOOb synthesis has been correlated with the arrest of cell Biol. Chem. 266, 7121-7131. proliferation and cell differentiation (27) and the protein has 24. Shaulsky, G., Ben-Ze'ev, A. & Rotter, V. (1990) Oncogene 5, been found maximally expressed in the G1 phase of the cell 1707-1711. 25. Whitfield, J. F., Sikorska, M., Boynton, A. L., Durkin, J. P., cycle (28), suggesting that it might contribute to the Ca2+ Kleine, L., Rixon, R. H. & Youdale, T. (1986) in Intracellular dependency of the G1 phase. SlOOb has been shown to Calcium Regulation, eds. Bader, H., Gietzen, K., Rosenthal, J., interact with most of the PKC substrates so far identified in Rudel, R. & Wolf, H. U. (Manchester Univ. Press, Manchester, brain tissues (12, 16, 29) and with p36 (calpactin I) (30), a U.K.), pp. 179-202. major substrate for the oncogene tyrosine kinase pp60src that 26. Kligman, D. & Hilt, D. C. (1988) Trends Biochem. Sci. 9, 437-443. is part of the associated with DNA 27. Zimmer, D. B. & Van Eldik, L. J. (1989) J. Cell Biol. 108, 141-151. protein complex poly- 28. Marks, A., Petsche, D., O'Hanlon, D., Kwong, P. C., Stead, R., merase a (31). It has therefore been suggested that the Dunn, R., Baumal, R. & Liao, S. K. (1990) Exp. Cell Res. 187, various demonstrated effects of the SlOOb proteins in cell 59-64. cycle progression and cell-type differentiation may, in fact, 29. Deloulme, J. C., Sensenbrenner, M. & Baudier, J. (1989) in Calcium be mediated by the same basic processes-i.e., interaction Binding Proteins in Normal and TransformedCells, eds. Pochet, R., with protein kinase substrates and modulation of target Lawson, D. E. & Heizmann, C. W. (Plenum, New York), pp. kinase substrate 153-157. protein phosphorylation (26, 29). The ob- 30. Hagiwara, M., Ochiai, M., Owada, K., Tanaka, T. & Hidaka, H. servation that SlOOb interacts with p53 in a calcium- (1988) J. Biol. Chem. 263, 6438-6441. dependent manner and that such an interaction inhibits p53 31. Jindal, H. K., Chaney, W. G., Anderson, C. W., Davis, R. G. & phosphorylation by PKC further supports this hypothesis. Vishwanatha, J. K. (1991) J. Biol. Chem. 266, 5169-5176. Downloaded by guest on September 28, 2021