Role of phosphorylation in p53 acetylation and PAb421 epitope recognition in baculoviral and mammalian expressed proteins Lorna J. Warnock, Sally A. Raines, Trevor R. Mee and Jo Milner YCR p53 Research Group, Department of Biology, University of York, York, UK In response to genotoxic stress, the p53 tumour sup- pressor protein is transiently stabilized, accumulates in the nucleus and acts as a transcription factor [1,2]. Sta- bilization of p53 occurs concomitantly with post-trans- lational modifications, which activate p53 for DNA repair, apoptosis and ⁄ or cell cycle arrest at G 1 ⁄ Sor G 2 ⁄ M [3,4]. p53 is post-translationally modified by phosphorylation, acetylation, ubiquitination, sumoyla- tion and neddylation [5–7]. Of these modifications, phosphorylation and acetylation have been studied in greatest detail [8]. Phosphorylation occurs at multiple sites within the N and C termini of p53 [9–11]. Acety- lation of p53 can occur at C-terminal lysines K320, K370-373, K381 and K382, but K373 and K382 appear to be the major sites for acetylation [12,13]. More recently, lysine 305 has also been identified as a novel p300-mediated p53 acetylation site [14]. In this work we generated a series of p53 ‘phos- phorylation’ mutants and constructed vectors for their expression either in insect cells, using baculoviral expression vectors (BVEs), or in a mammalian in vitro transcription ⁄ translation reticulocyte lysate system. Using antibodies specific for the modified residues of p53 we explored the influence of specific phosphoryla- tion mutants upon the actual phosphorylation of p53 at S15 and upon the acetylation of p53 at K373 and at K382. Results and Discussion His-tagged recombinant p53 proteins were expressed in Sf9 insect cells by using the baculoviral system and were purified as described previously [15]. Both human and murine p53 proteins were studied and included a Keywords acetylation; p53 protein; PAb421 epitope; phosphorylation Correspondence L. J. Warnock, YCR p53 Research Group, Department of Biology, University of York, York, YO10 5 DD, UK Fax: +44 1904 328622 Tel: +44 1904 328624 E-mail: ljw7@york.ac.uk (Received 1 December 2004, revised 24 January 2005, accepted 31 January 2005) doi:10.1111/j.1742-4658.2005.04589.x Post-translational modifications, such as phosphorylation and acetylation of the tumour suppressor protein p53, elicit important effects on the func- tion and the stability of the resultant protein. However, as phosphorylation and acetylation are dynamic events subject to complex controls, elucidating the relationships between phosphorylation and acetylation is difficult. In the present study we sought to address this problem by comparing full- length wild-type p53 with full-length p53 proteins mutated at specific phos- phorylation targets. Recombinant murine p53 proteins were expressed in insect cells (using the baculoviral expression vector system) and in a mam- malian in vitro transcription ⁄ translation reticulocyte lysate system. In p53 proteins derived from baculoviral expression vectors, S37A (but not S37D) was found to abrogate phosphorylation at S15. Lysine 382 (K382) is con- stitutively acetylated and was shown to form part of the epitope recognized by PAb421. Lysine 373 (K373) was only acetylated following substitutions at S315 (S315A or S315D) or at S378 (S378A). Importantly, in baculoviral expressed proteins, PAb421 reactivity was independent of K373 acetylation status, indicating that acetylation at K382 specifically determines the PAb421 epitope. Abbreviations BVE, baculoviral vector expression system. FEBS Journal 272 (2005) 1669–1675 ª 2005 FEBS 1669 dimeric mutant of murine p53 (M340Q ⁄ L344R), which can form dimers but not tetramers [16]. We have previ- ously characterized the quality of p53 protein obtained under the culture conditions employed here and dem- onstrated (a) wild-type conformational structure (react- ive with the mAb PAb1620) and (b) the formation of tetramers for intact p53 and also the formation of dimers for the dimeric mutant. There was little, if any, aggregation of the purified p53 protein (A. Okorokov & J. Milner, Dept Biology, University of York, unpub- lished data). Equivalent aliquots of p53 protein were resolved by SDS ⁄ PAGE and immunoblotted with site- specific monoclonal antibodies (mAbs), using PAb240 as reference control for total p53 (Fig. 1; note that PAb240 and anti-His-tag Ig gave equivalent results for p53 protein; data not shown). Serine 15 is constitutively phosphorylated and lysine 382 is constitutively acetylated First, we ascertained the constitutive status of p53 modifications at serine 15 (S15) and at lysine 373 (K373) and lysine 382 (K382) for recombinant p53 expressed in insect cells. The results clearly show that S15 is constitutively phosphorylated on wild-type p53, both human and murine, and also on a dimeric mutant of murine p53 (M340Q ⁄ L344R, Fig. 1). In the case of acetylation we observed site-specific differences at K373 and K382. Thus there was little, if any, acetyla- tion at K373 (K373Ac), whereas K382 appeared to be constitutively acetylated. These findings are consistent with other publications which have reported that phos- phorylation at serine 15, a modification that occurs in response to genotoxic stress, enhances the binding of p53 to p300 ⁄ CBP and increases the level of acetylation at lysines 320 and 382 [10,17,18]. Note that, although the anti-K373Ac ⁄ K382Ac Ig detects both K373Ac and K382Ac, we interpreted a positive signal to be specific for K382Ac when K373 was shown to be nonacetylated by the site-specific anti-K373Ac serum (as in Fig. 2). From these results we conclude that both human and murine wild-type p53 are constitutively phosphorylated at S15 and acetylated at K382, but not at K373. Next, we tested a series of murine p53 phosphoryla- tion mutants, expressed in the baculoviral system, for any effects on the constitutive pattern of p53 modifica- tions at S15 and at K373 and K382. The results are shown in Fig. 2 and summarized in Table 1. Several points of interest emerge and are discussed below. S37A abrogates p53 phosphorylation at S15 It is apparent that the substitution of alanine at S37 completely abrogates the constitutive phosphorylation at S15 (Fig. 2, S37A, row 2, lane 5). In contrast, S37D has no effect on S15 phosphorylation (Fig. 2, S37D, PAb240 S15 P K373Ac K373/K382Ac Mouse WT Dimer Human WT Fig. 1. Recombinant human and murine wild-type p53 proteins are phosphorylated at S15 and acetylated at K382 when expressed in insect cells. Western blot analyses of purified p53 proteins are shown, using PAb240 as reference control for total p53 protein. Phosphorylation at S15 was detected by using phospho-p53 (S15) antibody (New England Biolabs). Acetylated K373 was detected by anti-(acetyl p53 lysine 373) Ig (Abcam). Acetylated K373 and ⁄ or K382 were detected by anti-(acetyl-p53 lysine 373 ⁄ 382) Ig (Up- state). Peroxidase chemiluminescence blotting was performed in accordance with the manufacturer’s instructions (Roche Diagnos- tics). S15P WT Dimer S15A S15D S37A S37D S315A S315D S376A S376D S378A S378D S376D/S378D 1 234 5678910 111213 PAb240 K373Ac K373/K382Ac Fig. 2. Phosphorylation status at S15 and acetylation status at K373 and K382 of murine wild-type p53 and p53 proteins mutated at specific phosphorylation sites expressed in insect cells by using PAb240 as a reference control for total p53 protein. Western blot analyses of murine full-length p53 proteins. Lane 1, murine wild- type p53; lane 2, dimer mutant protein (M340Q ⁄ L344R); lanes 3–13, phosphorylation mutant proteins, as indicated. p53 phosphorylation, acetylation and PAb421 epitope L. J. Warnock et al. 1670 FEBS Journal 272 (2005) 1669–1675 ª 2005 FEBS row 2, lane 6). Other workers have reported that the removal of S15 abrogates phosphorylation at S9, T18 and S20 [19]. These findings are consistent with the theory of S15 cluster phosphorylation site interdepend- ency. Given the well-established importance of p53 S15P in the cellular response to DNA damage, the discovery that S37 modification may be regulatory for S15 phosphorylation in baculoviral-expressed proteins remains highly significant. As expected, S15A was nonreactive with anti-(phos- phoserine 15) Ig (Fig. 2, row 2, lane 3), marking a loss of ability to phosphorylate the substituted alanine at this site. In contrast, S15D was still detected by anti- (phosphoserine 15) Ig (Fig. 2, row 2, lane 4), confirm- ing that the aspartic acid substitution mimics the phosphorylation ‘status’ of the epitope. None of the mutations at S15 and S37 (S15A, S15D, S37A or S37D) affected the acetylation profile of p53 protein at K373 (Fig. 2, row 3, lanes 3–6) or at K382 (Fig. 2, bottom row, lanes 3–6). These results are supported by other workers who found that following the deletion of p53 N-terminal residues 13–52, proteins expressed in MEFs remained acetylated at K382 [20] [Fig. 2, note that phospho-p53 (S15) antibody (New England Biolabs) and p-p53 (Ser 15)-R antibody (Santa Cruz Biotechnology, Inc.) gave equivalent results for p53 protein. Results shown are those achieved with the former antibody.] Substitution at S315 induces p53 acetylation at K373 S315 is juxtaposed to the major nuclear localization sig- nal of p53 and its phosphorylation involves S and G 2 ⁄ M cyclin-dependent kinases [21]. Our results now indicate that in protein derived from BVEs, an intact serine resi- due at this site has important ‘long-range’ effects on p53 post-translation modification as both alanine and aspar- tic acid substitutions at S315 result in the acetylation of K373 (Fig. 2, row 3, lanes 7 and 8). It was not possible to determine formally whether K382 was acetylated or de-acetylated under these conditions because anti- K382Ac Ig also detects K373Ac (see above). Modifications at S376 and S378 differentially affect p53 acetylation For proteins expressed in BVEs, our results indicate that serines 376 and 378 also impact upon acetylation at K373 and K382. Substitution of either alanine or aspartic acid at S376 resulted in the loss of acetylation at K382 (Fig. 2, S376A and S376D, bottom row, lanes 9 and 10). This site is normally constitutively acetylat- ed under these experimental conditions (Fig. 1) and we conclude that S376 may be structurally important for acetylation to occur at K382 in the native p53 protein. A different picture of p53 acetylation is evident for phosphorylation mutants at S378. Here, the aspartic acid substitution, S378D, abolishes constitutive acety- lation at K382 without effect on K373 status (Fig. 2, bottom row, lane 12). Results from the dual-phos- phorylation mutant protein, S376D ⁄ S378D, further support this data (Fig. 2, bottom row, lane 13). This finding is important as it has recently been shown that acetylation at K382 is responsible for p53-induced p21 activation and cell growth arrest in G 1 [20]. The alan- ine substitution, S378A, however, resulted in a positive signal for K373Ac (Fig. 2, bottom row, lane 11). Thus, the results obtained for S378A are similar to those obtained for S315A ⁄ D (see above). S378A appears to facilitate the selective acetylation of K373 in p53. K382Ac is part of the PAb421 epitope Phosphorylation and acetylation cascades have many aspects in common and are linked in the regulation and function of p53 [4,5,22]. The interplay between phosphorylation and acetylation of p53 at residues K373 to K382 is very interesting. These same residues encompass the epitope for the mAb PAb421, once believed to activate p53 [23,24]. We were curious to determine whether any of the C-terminal p53 modi- fications, described above, affect epitope recognition Table 1. Summary of p53 S15 phosphorylation status, K373 and K382 acetylation status, and immunoreactivity with PAb421 and PAb122 for the full-length p53 variants derived from the baculoviral expression vector system (BEV) system used in this study. PAb240 S15P K373Ac K373 ⁄ K382Ac PAb421 PAb122 Hwt +++ +++ Mwt ++- +++ Dimer + + - + + + S15A + - - + + + S15D + + - + + + S37A + - a -+++ S37D + + - + + + S315A + + + + + + S315D + + + + + + S376A + + - - - - S376D + + - - - - S378A + + + + + a + S378D + + - - - - S376D ⁄ S378D + + - - - - a Note that, in contrast to the BVE system, S37A protein derived from the mammalian in vitro transcription ⁄ translation reticulocyte lysate was immunoreactive with phospho-p53 (S15) antibody, and mammalian expressed S378A protein was not immunoreactive with PAb421. L. J. Warnock et al. p53 phosphorylation, acetylation and PAb421 epitope FEBS Journal 272 (2005) 1669–1675 ª 2005 FEBS 1671 by PAb421. A set of clear-cut results for baculoviral proteins emerged. All p53 mutants that lacked acetyla- tion at K382 (namely S376A, S376D, S378D and the double mutant, S376D ⁄ S378D; Fig. 2) also failed to react with PAb421 (Fig. 3A, row 2 and Table 1). The same was true for reactivity with PAb122, an anti-p53 mAb which detects an epitope similar, but not identi- cal to, PAb421 (Fig. 3A, row 3 and Table 1). Thus, we conclude that for proteins expressed in the baculoviral system, acetylation at K382 is required for reactivity with the PAb421 epitope. Our present observations allow further refinement of the PAb421 epitope to include K382Ac. Acetylation at K382 is site-specific for PAb421 reactivity, as reactivity was unaffected by the acetylation status of K373 (see Table 1). Our results also indicate that acetylation of K382 coupled with PAb421 reactivity are subject to modifications at the neighbouring serine residues, S376 and S378, which may thus play important (albeit indirect) roles in regu- lating p53 functions [25,26]. The observation that acetylation at K382 (K382Ac) forms part of the epitope for PAb421 is new and important. In order to verify this hypothesis we expan- ded our study to include bacterially expressed p53 proteins because, in the bacterial system, post-transla- tional modifications (including phosphorylation and acetylation) are deficient. Recombinant murine wild-type p53 protein was expressed in Escherichia coli BL21 cells (see the Experi- mental procedures). Equivalent aliquots of p53 protein were resolved by SDS ⁄ PAGE and immunoblotted with site-specific mAbs, using PAb240 as reference control for total p53 (Fig. 3B). We determined the status of modifications at S15, following p53 expression in E. coli, by using phospho- p53 (S15) antibody (New England Biolabs) or p-p53 (Ser 15)-R antibody (Santa Cruz Biotechnology, Inc.). As expected, we were unable to detect phosphorylation at S15 with either antibody. In addition, murine wild- type p53 protein expressed in E. coli was nonreactive with PAb421 (Fig. 3b). This observation supports our conclusion that acetylation at K382 is necessary in for- mation of the PAb421 epitope (see above). In previous studies, human wild-type p53 protein expressed in E. coli was shown to be reactive with PAb421 [4]. A possible explanation for variable PAb421 reactivity is the fact that availability of the PAb421 epi- tope is affected by phosphorylation [25] and acetylation (see above). Wild-type murine p53 protein, when expressed in E. coli BL21 in this study, was found to exhibit little, if any, PAb421 reactivity relative to PAb240 (Fig. 3B). However, it is effective for DNA binding with some PAb421 reactivity, as revealed by gel shifts (data not shown). Our data from the BVE system led us subsequently to ascertain the constitutive status of p53 modifications at S15 and at K373 and K382 for recombinant p53 expressed in a mammalian in vitro transcription ⁄ trans- lation reticulocyte lysate system. p53 proteins are expressed in the wild-type conformation and S15 is constitutively phosphorylated Equivalent aliquots of p53 protein derived from the mammalian in vitro transcription ⁄ translation reticulo- cyte lysate system were immunoprecipitated with site- specific mAbs and resolved by SDS ⁄ PAGE. The p53 proteins tested were found to have wild-type confor- mation (Fig. 4, lanes 2–5) and are constitutively phos- phorylated at S15 (Fig. 4, lane 8). In contrast to the BVE system, our results showed that substitution of an alanine at S37 did not abrogate WT Dimer S15A S15D S37A S37D S315A S315D S376A S37 6D S378A S37 8D PAb240 PAb122 S376D/S378D PAb421 S15P BVE WT PAb240 PAb421 E. Coli WT A B Fig. 3. Monoclonal antibodies PAb421 and PAb122 are nonreactive with the baculoviral vector expression system (BEV)-expressed murine p53 phosphorylation mutant proteins, which are deacety- lated at lysine 382. (A) Immunoreactivity of p53 proteins with PAb421 and PAb122. Lane 1, murine wild-type p53; lane 2, dimer mutant protein (M340Q ⁄ L344R); lanes 3–13, phosphorylation mutant proteins, as indicated. PAb240 was used as a reference control for total p53 protein. (B) Comparison of the immunoreactivi- ty of purified murine wild-type p53 proteins with phospho-p53 (S15) antibody and PAb421 expressed in BVE or E. coli BL21-competent cells. Western blot analysis of p53 protein using PAb240 as a refer- ence control for total p53 protein. p53 phosphorylation, acetylation and PAb421 epitope L. J. Warnock et al. 1672 FEBS Journal 272 (2005) 1669–1675 ª 2005 FEBS the constitutive phosphorylation at S15 (Fig. 4, row 2, lane 8). Thus, we conclude that for specific site recog- nition, kinases responsible for phosphorylation of S15 in insect cells are more stringent in their requirement for phosphorylation at S37. C-terminal p53 modifications inhibit PAb421 epitope recognition Wild-type, S37A and S37D proteins derived from the mammalian in vitro transcription ⁄ translation reticulo- cyte lysate system were reactive with PAb421. How- ever, C-terminal p53 modifications inhibited epitope recognition by PAb421 (Fig. 4, lane 6). This is consis- tent with studies in a mammalian system which repor- ted that C-terminal phosphorylation by protein kinase C abolishes recognition of the PAb421 epitope [25]. We conclude that the bacterial, mammalian and BVE protein expression systems may differ in their post- translational modifications, leading to differences in availability of the PAb421 epitope. Overall, our results are consistent with the view that phosphorylation and acetylation occur in concert, the dynamics of which are dependent upon the environment in which proteins are expressed and post-translationally modified [8]. Experimental procedures Baculoviral expression and purification of p53 proteins Phosphorylation mutant p53 proteins were generated by site-directed mutagenesis [27]. Using murine p53 cDNA as a template, mutation constructs of recombinant baculovirus encoding His-tagged p53 derivatives were produced as pre- viously described [28]. The human numbering of residues for p53 was used throughout this study. Target genes were subcloned into the vector pVL 1393 (Pharmingen, San Diego, CA, USA) for expression in the baculoviral system and were then integrated into the baculoviral genome by using the BaculoGold TM System (PharMingen). The resul- tant recombinant His-tagged proteins were expressed and purified to homogeneity, as previously described [15]. Pro- tein concentration was quantified by using the Bradford Assay (BioRad, Hercules, CA, USA). Western blot analysis Western blot analysis was performed as described previ- ously [29]. PAb240 from hybridoma cell lines detected the presence of the epitope comprising amino acids 212–217 of the p53 protein and represents the amount of p53 protein loaded in each lane [30]. Phosphorylation at S15 was detec- ted by using phospho-p53 (S15) antibody (New England Biolabs, Beverley, MA, USA) or p-p53 (Ser 15)-R antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), both of which yielded equivalent results. Acetylated K373 was detected by anti-acetyl p53 lysine 373 (Abcam, Cambridge, MA, USA). Acetylated K373 and ⁄ or K382 were detected by anti-acetyl-p53 lysine 373 ⁄ 382 (Upstate Charlottesville, VA, USA). PAb421 and PAb122 from hybridoma cell lines detected the presence of the epitopes comprising amino acids 371–382 and amino acids 370–378 of the p53 protein, respectively [31]. Peroxidase chemilumi- nescence blotting was performed in accordance with the manufacturer’s instructions (Roche, Mannheim, Germany). Expression and purification of murine wild-type p53 protein in E. coli Using murine wild-type p53 cDNA as a template, con- structs encoding a His-tagged p53 derivative were cloned into the vector pcDNA3, and plasmids were transformed into E. coli BL21 competent cells (Novagen). The protein was induced by 1 mm isopropyl thio- b -d-galactoside at WT Total Lysate 248 246 1620 240 421 419 S15 S37A S376A S376D S378A S378D S37D 12345678 Fig. 4. p53 proteins derived from the mammalian in vitro transcrip- tion ⁄ translation reticulocyte lysate system showed wild-type p53 conformation and constitutive phosphorylation at S15. Resultant radiolabelled proteins were immunoprecipitated with PAb419 as a negative control (lane 7). Extracts were analysed by SDS gel elec- trophoresis and autoradiography. L. J. Warnock et al. p53 phosphorylation, acetylation and PAb421 epitope FEBS Journal 272 (2005) 1669–1675 ª 2005 FEBS 1673 37 °C for 4 h and extracted with 4· Laemelli buffer [8% (w ⁄ v) sodium dodecyl sulfate, 40% (v ⁄ v) glycerol, 400 mm dithiothreitol, 240 mm Tris, pH 6.8, and 0.001% (w ⁄ v) bromophenol blue]. In vitro transcription, translation and immunoprecipitation In vitro transcription, translation and immunoprecipitation in the mammalian rabbit reticulocyte lysate system were performed as described previously [27]. pGEM plasmids encoding the wild-type or mutant p53 gene under the SP6 promoter were incorporated. The resultant radiolabelled proteins were immunoprecipitated with PAb419 as a negat- ive control [31]. mAbs PAb248 (41–52), PAb246 (202–204), PAb1620 (156, 206, 209, 210), PAb240 (212–217) and PAb421 (371–382) were used to determine the conforma- tional status of the proteins [30]. Phosphorylation at S15 was detected by using phospho-p53 (S15) antibody (New England Biolabs). Acknowledgements We thank Dr Rachel Adamson, Dr Jack Ford and Dr Carlos Rubbi for critical reading of the manuscript. This work was supported by a Yorkshire Cancer Research program grant awarded to Professor Jo Milner. 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Mee and Jo Milner YCR p53. recombinant p53 proteins were expressed in Sf9 insect cells by using the baculoviral system and were purified as described previously [15]. Both human and murine p53 proteins were studied and included. 111213 PAb240 K373Ac K373/K382Ac Fig. 2. Phosphorylation status at S15 and acetylation status at K373 and K382 of murine wild-type p53 and p53 proteins mutated at specific phosphorylation sites expressed in insect cells by using PAb240