TP53INP1s and Homeodomain-interacting Protein Kinase-2 (HIPK2) Are Partners in Regulating p53 Activity

Abstract

The TP53INP1 gene encodes two protein isoforms, TP53INP1α and TP53INP1β, located into the nucleus. Their synthesis is increased during cellular stress by p53-mediated activation of transcription. Overexpression of these isoforms induces apoptosis, suggesting an involvement of TP53INP1s in p53-mediated cell death. It was recently shown that p53-dependent apoptosis is promoted by homeodomain-interacting protein kinase-2 (HIPK2), which is known to bind p53 and induce its phosphorylation in promyelocytic leukemia protein nuclear bodies (PML-NBs). In this work we show that TP53INP1s localize with p53, PML-IV, and HIPK2 into the PML-NBs. In addition, we show that TP53INP1s interact physically with HIPK2 and p53. In agreement with these results we demonstrate that TP53INP1s, in association with HIPK2, regulate p53 transcriptional activity on p21, mdm2, pig3, and bax promoters. Furthermore, TP53INP1s overexpression induces G1 arrest and increases p53-mediated apoptosis. Although a TP53INP1s and HIPK2 additive effect was observed on apoptosis, G1 arrest was weaker when HIPK2 was transfected together with TP53INP1. These results indicate that TP53INP1s and HIPK2 could be partners in regulating p53 activity. The TP53INP1 gene encodes two protein isoforms, TP53INP1α and TP53INP1β, located into the nucleus. Their synthesis is increased during cellular stress by p53-mediated activation of transcription. Overexpression of these isoforms induces apoptosis, suggesting an involvement of TP53INP1s in p53-mediated cell death. It was recently shown that p53-dependent apoptosis is promoted by homeodomain-interacting protein kinase-2 (HIPK2), which is known to bind p53 and induce its phosphorylation in promyelocytic leukemia protein nuclear bodies (PML-NBs). In this work we show that TP53INP1s localize with p53, PML-IV, and HIPK2 into the PML-NBs. In addition, we show that TP53INP1s interact physically with HIPK2 and p53. In agreement with these results we demonstrate that TP53INP1s, in association with HIPK2, regulate p53 transcriptional activity on p21, mdm2, pig3, and bax promoters. Furthermore, TP53INP1s overexpression induces G1 arrest and increases p53-mediated apoptosis. Although a TP53INP1s and HIPK2 additive effect was observed on apoptosis, G1 arrest was weaker when HIPK2 was transfected together with TP53INP1. These results indicate that TP53INP1s and HIPK2 could be partners in regulating p53 activity. The stress-induced protein (SIP) gene, recently cloned by Tomasini et al. (1Tomasini R. Samir A.A. Vaccaro M.I. Pebusque M.J. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2001; 276: 44185-44192Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), is strongly activated in the diseased pancreas. The mouse SIP gene expands over ∼20 kb and is organized in 5 exons. Exon 4 is alternatively spliced to generate two transcripts that encode proteins of 18 and 27 kDa (SIP18 and SIP27). SIP27 is identical to the TEAP protein, formerly described as a thymus-expressed protein (2Carrier A. Nguyen C. Victorero G. Granjeaud S. Rocha D. Bernard K. Miazek A. Ferrier P. Malissen M. Naquet P. Malissen B. Jordan B.R. Immunogenetics. 1999; 50: 255-270Crossref PubMed Scopus (60) Google Scholar). SIP18 and SIP27 are localized mainly in the nucleus, as shown in previous experiments using SIP-GFP fusion proteins (1Tomasini R. Samir A.A. Vaccaro M.I. Pebusque M.J. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2001; 276: 44185-44192Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In vitro, expression of SIP mRNAs is strongly induced in fibroblasts submitted to various stress agents such as UV, base damage, ethanol, heat shock, and oxidative stress. It is noteworthy that mRNAs encoding SIP18 and SIP27 are concomitantly activated in response to these stresses. We also showed that SIP gene expression is p53-dependent (3Tomasini R. Samir A.A. Pebusque M.J. Calvo E.L. Totaro S. Dagorn J.C. Dusetti N.J. Iovanna J.L. Eur. J. Cell Biol. 2002; 81: 294-301Crossref PubMed Scopus (77) Google Scholar). Finally, we found that overexpression of SIP18 or SIP27 induces apoptosis, indicating that SIP gene expression is a key element in p53-mediated cell death that occurs as a consequence of cellular stress. Independently, Okamura et al. (4Okamura S. Arakawa H. Tanaka T. Nakanishi H. Ng C.C. Taya Y. Monden M. Nakamura Y. Mol. Cell. 2001; 8: 85-94Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) cloned a new human gene named p53DINP1. This gene encodes a p53-inducible nuclear protein that promotes apoptosis when overexpressed in MCF7 cells. Sequence analysis revealed that SIP, p53DINP1, and TEAP are in fact alternative names for the same gene that was approved as TP53INP1 (tumor protein 53-induced nuclear protein 1) by the HUGO Gene Nomenclature Committee, with TP53INP1α and TP53INP1β proteins corresponding to SIP18 and SIP27, respectively. Okamura et al. (4Okamura S. Arakawa H. Tanaka T. Nakanishi H. Ng C.C. Taya Y. Monden M. Nakamura Y. Mol. Cell. 2001; 8: 85-94Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) demonstrated, using a very elegant approach, that TP53INP1s bind to a kinase that phosphorylates p53 at Ser-46, such a phosphorylation being apparently crucial for UV-induced apoptosis (5Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (598) Google Scholar, 6Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar). Unfortunately, that critical kinase was not identified in Okamura's work. More recently, two papers showed that in subnuclear structures called promyelocytic leukemia protein nuclear bodies (PML-NBs), 1The abbreviations used are: PML, promyelocytic leukemia protein; NBs, nuclear bodies; GFP, green fluorescence protein; PIG3, p53-inducible gene-3; TRITC, tetramethylrhodamine isothiocyanate; wt, wild type; HIPK2, homeodomain-interacting protein kinase-2; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; CMV, cytomegalovirus. the homeodomain-interacting protein kinase-2 (HIPK2) binds to p53 and phosphorylates its Ser-46 to promote p53-dependent apoptosis (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar). Taken together, these data suggest that HIPK2 could be the kinase that binds to TP53INP1s. The aim of the present work was to investigate a possible relationship between TP53INP1s, HIPK2, and p53. Cell Culture and Transfections—HEK 293, MEF p53–/–, RasV12/E1A-transformed MEFs (9Vasseur S. Hoffmeister A. Garcia S. Bagnis C. Dagorn J.C. Iovanna J.L. EMBO Rep. 2002; 3: 165-170Crossref PubMed Scopus (69) Google Scholar), RKO and U2OS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen), 1% (w/v) penicillin/streptomycin, in a 5% CO2 incubator at 37 °C. Cells were transfected with FuGENE-6 reagent (Roche Applied Science) in accordance with the manufacturer's instructions. RasV12/E1A-transformed MEFs were irradiated with γ-rays (30 gray) using a Gammacell 1000 radiator. Antibodies and Plasmids—Antibodies recognizing FLAG (F7425) and β-tubulin (TUB 2.1) were from Sigma; p53 (FL-393), PML (PM-G3), anti-p21WAF1 (sc-397), and goat polyclonal anti-rabbit IgG rhodamine (SC-2091) were from Santa Cruz Biotechnology Inc. Goat polyclonal anti-mouse IgG TRITC-conjugated and goat polyclonal anti-rabbit IgG Cy-5 were from Jackson Immunoresearch; anti-V5 (46–0705) was from Invitrogen; monoclonal antibody TP53INP1 (anti-E12) was generated in our laboratory. 2M. J. Xie, A. Carrier, S. Garcia, A. Azizi Samir, J. L. Iovanna, and M. J. Pébusque, submitted for publication. pGST-TP53INP1α and pGST-TP53INP1β, pGST-HIPK2-(1–1189), pGST-HIPK2-(1–853), pGST-HIPK2-(853–955), pGST-p53-(13–390), and pGST-p53-(295–390) were constructed in the pGEX plasmid (Amersham Biosciences) (Fig. 1a). The control plasmid pCMV/βgal was from Promega. pHIPK2-FLAG (8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar), pLHIPK2SP and pLxSP (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar), pTP53INP1α-V5, pTP53INP1β-V5, pTP53INP1α-GFP, and pTP53INP1β-GFP (1Tomasini R. Samir A.A. Vaccaro M.I. Pebusque M.J. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2001; 276: 44185-44192Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) as well as pPML-IV and pPML-L (10Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (321) Google Scholar) were described previously. pcDNA-Hu-p53wt plasmid was a kind gift from A. Sparks (University of Dundee, United Kingdom). In Vitro Pull-down and Co-immunoprecipitation Assays—GST fusion proteins were produced in Escherichia coli BL21 and purified on glutathione-Sepharose resin (Amersham Biosciences). Proteins translated in vitro were synthesized with a TnT-coupled reticulocyte lysate system in the presence of l-[35S]methionine according to the manufacturer's instructions (Promega). Labeled proteins were incubated with 10 μg of GST fusion protein at 4 °C for 2 h with in vitro interaction buffer (50 mm Hepes, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% (v/v) glycerol, 1% Triton X-100, and a mix of protease inhibitors), washed four times with 500 μl of in vitro interaction buffer, and two times with 500 μl of PBS. Bound proteins were eluted with 1× SDS sample buffer, separated by denaturing SDS-PAGE, and analyzed by autoradiography. For in vivo co-immunoprecipitation, U2OS cells were plated on 60-mm Petri dishes and transiently transfected. Thirty-six hours after transfection, total cellular extracts were prepared with lysis buffer (50 mm Tris HCl, pH 7.5, 5 mm EDTA, 300 mm NaCl, 150 mm KCl, 1 mm dithiothreitol, 1% Nonidet P40, and a mix of protease inhibitors). Equal amounts of proteins were immunoprecipitated for 2 h at 4 °C with 10 μl/sample of anti-V5 monoclonal antibody (Invitrogen) on protein A-agarose beads (Roche Applied Science). Immunocomplexes were collected by centrifugation, washed five times with lysis buffer, separated by SDS-PAGE, and blotted onto nitrocellulose membranes (Amersham Biosciences). Filters were blocked in Tris-buffered saline-Tween 0.1% plus 3% dried skimmed milk and incubated with anti-FLAG monoclonal antibody or anti-p53 rabbit polyclonal antiserum. Immunofluorescence—Cells were grown on coverslips in 6-well plates and transfected with 2 μg of expression vector. Twenty-four hours later they were washed once with 1× PBS, fixed for 15 min at room temperature in PBS-3% paraformaldehyde, and then washed twice with PBS and blocked in PBS, 50 mm NH4Cl for 10 min at room temperature. Cells were then incubated for 60 min with the primary antibodies at room temperature and washed four times (5 min each) in PBS before incubation for 60 min with the appropriate fluorochrome-conjugated secondary antibodies. Cells were mounted on glass slides and examined with a confocal laser microscope (TCS NT Leica apparatus, Heidelberg, Germany). Trans-activation Assays—Cells were transiently transfected with reporter vectors in which the luciferase gene was driven by the p53-dependent intronic promoter of the murine mdm2 gene (11Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1181) Google Scholar), the human bax promoter (12Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar), the pig3 promoter (13Polyak K. Xia Y. Zweir J.L. Winzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar), or the p21WAF1 promoter (kindly provided by M. Oren and B. Vogelstein). Transcriptional activity was evaluated after co-transfection with the indicated expression constructs. Twenty-four hours after transfection, cellular extracts were analyzed for luciferase activity and results were normalized to β-galactosidase activity. All were with vector. analysis of μg of total cellular extracts were separated by SDS-PAGE, blotted onto (Amersham and incubated with anti-p21WAF1 monoclonal antibody and for Filters were washed in Tris-buffered saline-Tween and then incubated with Antibodies were with an (Amersham Biosciences) in accordance with the manufacturer's instructions. Cell were transiently transfected with the indicated expression constructs. The they were fixed in for Cells were with 18 for 15 min at room temperature in the presence of cells were in the of a Biosciences) with Their cellular was with a in the cells were plated into Petri dishes and transfected h later with the indicated expression vectors using FuGENE-6 reagent as described The transfected cells were in and for 10 and with to the of was Cell death was evaluated in cells transfected with pTP53INP1α-GFP, or pTP53INP1β-GFP or in with Cells were plated on glass coverslips for h to h after transfection, cells or fusion proteins were for of apoptosis by and fluorescence the of green cells this on were This was in TP53INP1s HIPK2 and was to TP53INP1s bind to this in vitro experiments were to demonstrate a interaction between and with shown in and bind to the HIPK2, HIPK2 not bind to the control GST we also found that and bind to p53 (Fig. These in vitro were by experiments using and together with TP53INP1α and TP53INP1β (Fig. that TP53INP1α and TP53INP1β also interact with HIPK2 and p53 in we cells with with or p53 constructs. shown in bind to p53 and was used to induce a stress in RasV12/E1A-transformed which are known to TP53INP1 expression (4Okamura S. Arakawa H. Tanaka T. Nakanishi H. Ng C.C. Taya Y. Monden M. Nakamura Y. Mol. Cell. 2001; 8: 85-94Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, S. Hoffmeister A. Garcia S. Bagnis C. Dagorn J.C. Iovanna J.L. EMBO Rep. 2002; 3: 165-170Crossref PubMed Scopus (69) Google Scholar). Twenty-four hours after cells were collected and and total cellular extracts were immunoprecipitated with antibodies. shown in p53 and HIPK2 are with Taken together, these results demonstrate that overexpressed and proteins interact with HIPK2 and p53 in vitro and in We experiments to the which TP53INP1α and bind to HIPK2 and p53. shown in TP53INP1α and TP53INP1β bind to and to the in the of It is known that the to of HIPK2 binds to the of p53 (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar). a between TP53INP1s and HIPK2 for p53 is the of p53 as a 2002; PubMed Scopus Google Scholar) to which proteins bind D. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction between TP53INP1s, HIPK2 and p53 we in the present suggest that could be in a TP53INP1s with HIPK2 and p53 into the of TP53INP1β, HIPK2, and p53 were It was recently that PML-IV, a PML p53 into structures named PML nuclear bodies (10Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (321) Google Scholar). This promotes p53 which its activity P. Pandolfi P.P. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are cell nuclear structures that as in A. 1999; PubMed Scopus Google Scholar). leukemia protein, the of these a in S. S. S. A. Pandolfi P.P. 2000; PubMed Google Scholar). The possible of in cell control is by in PML that indicate and M. J. J.C. The H. Nat. PubMed Scopus Google Scholar, D. S. S. M. R. Pandolfi P.P. Nat. PubMed Scopus Google Scholar). in the control of gene expression S. P. Pandolfi P.P. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar) and as a by G1 cell arrest S. M.C. PubMed Scopus Google Scholar). HIPK2 is also localized into the with (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar). We show that TP53INP1α and TP53INP1β are also by into the and with HIPK2 and p53 (Fig. a and to PML-IV, of PML, is to HIPK2 and p53 into the (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (321) Google Scholar). We that was also to TP53INP1α and TP53INP1β not by of TP53INP1s or HIPK2 the to be of p53 occurs in MEFs not Finally, control experiments using MEF nuclear revealed a for nuclear bodies and TP53INP1s. overexpression of in MEF also induces a TP53INP1s into the (Fig. Taken together, these results demonstrate that p53, HIPK2, and TP53INP1α and TP53INP1β into PML-NBs. The between these proteins be or promoted by such a which also the its of TP53INP1s and HIPK2 p53 Trans-activation p53 is a protein, and maintained at a and C. PubMed Scopus Google Scholar). p53 is activated at the by a of phosphorylation and of and A.J. 12: PubMed Scopus Google Scholar, Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). p53 activation to the of of which the a of such as cell apoptosis, or Oren M. PubMed Scopus Google Scholar). control such a of the p53 on be on the promoter of and cellular R. K. M. Y. D. E. A.J. 2000; PubMed Scopus Google Scholar). We suggest that TP53INP1s are in such a to HIPK2 and p53 with to p53-dependent we used MEFs as a to p53 Their together with a and a plasmid the luciferase reporter gene by of the the of p53 transcriptional activity. MEFs were transfected with vectors or or in with 4 that HIPK2 increased p53 transcriptional activity as (8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar). TP53INP1α or TP53INP1β also increased p53 activity effect was observed not TP53INP1 with HIPK2 in a of p53 activity indicating a results were using and cells not We that TP53INP1s regulate p53 transcriptional activity and that HIPK2 in this this we experiments using the luciferase reporter gene the control of mdm2, and of the (Fig. p53 activation of p21, mdm2, and bax was increased to various by TP53INP1α and TP53INP1β, to the pig3 which was not The was the in which p53-dependent expression was increased by TP53INP1α and by HIPK2 not p53 transcriptional activity on and mdm2 the TP53INP1 of and that of mdm2 p53-mediated activation of the bax promoter was increased by TP53INP1s and HIPK2 on Finally, TP53INP1 which not the p53 activity on the pig3 its activation by results were using cell such as and not a we by the expression of protein in MEF was increased by TP53INP1α and by TP53INP1β, HIPK2 expression such activation (Fig. These results are in agreement with our results from the experiments described is to that with in a of by p53 (Fig. of p53 transcriptional activity by TP53INP1s is MEFs were with a luciferase reporter gene by of the mdm2 pig3 and bax and of the expression or pTP53INP1β-V5, and were normalized to β-galactosidase activity. gene activation induced by pcDNA-Hu-p53wt was at are as for at of p21WAF1 expression by TP53INP1s is blocked by MEFs were transfected with of pTP53INP1α-V5, pTP53INP1β-V5, and pHIPK2-FLAG as p21WAF1 and were then by was used as a these data indicate that p53 transcriptional activity on is by HIPK2 and TP53INP1s. The and of the be on the promoter used not on the cell of TP53INP1s and HIPK2 strongly p53 activity. in p53 on be in to of TP53INP1s and TP53INP1s and HIPK2 in p53-mediated of Cell and results show that TP53INP1s and HIPK2 regulate the p53 activity on in cell and and apoptosis and We analyzed by the of overexpression in cells on the cell and apoptosis (Fig. a cells were transfected with expression with or pTP53INP1β-GFP in a in the of cells in G1 and in control indicating a G1 cell In agreement with previous results (1Tomasini R. Samir A.A. Vaccaro M.I. Pebusque M.J. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2001; 276: 44185-44192Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, S. Arakawa H. Tanaka T. Nakanishi H. Ng C.C. Taya Y. Monden M. Nakamura Y. Mol. Cell. 2001; 8: 85-94Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), we also found a in the of cells in to and with increased apoptosis. p21, in which p53-mediated activation is by TP53INP1s could be of the in the in G1 arrest Tokino T. R. D. K.W. Vogelstein B. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). Overexpression of HIPK2 also arrest to as (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (503) Google Scholar). In with TP53INP1s, HIPK2 produced a and in the of cells in to as well as a of cells in G1 to increased in was that was not by the (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (578) Google Scholar, M. G. R. G. M. L. A. 2001; PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, when of the TP53INP1s was overexpressed the of cells in G1 was when HIPK2 was the of cells in was increased suggesting a between the two proteins in the of p53 activity. the of TP53INP1s and HIPK2 on apoptosis were results in the cell and apoptosis were found in mouse embryo fibroblasts not suggesting that the of TP53INP1s with HIPK2 is not cell We used RKO which expression of or or in with Their activity was by a RKO cells were transfected with pLxSP and shown in cells transfected with the the the of by cells or was of was of or plasmid in with cells were also transfected with or pTP53INP1β-GFP or in with or with the expression plasmid as a hours after transfection, cells were analyzed by and fluorescence for of apoptosis and nuclear shown in the of cells of apoptosis was in cells transfected with or pTP53INP1β-GFP in control cells. The cell was by data not these data suggest that TP53INP1s and HIPK2 p53 activity and a in cell and apoptosis. We M. L. Schmitz for pHIPK2-FLAG M. for and pPML-L M. for MEFs p53–/–, S. for RasV12/E1A-transformed and R. and P. for are to for in analysis and M. J. and A. for critical of the