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• W2014102011 abstract "Protein kinase C (PKC) δ is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCδCF) in the apoptotic response of cells to DNA damage. Expression of PKCδCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCδCF associates with p73β, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCδCF phosphorylates the p73β transactivation and DNA-binding domains. One PKCδCF-phosphorylation site has been mapped to Ser-289 in the p73β DNA-binding domain. PKCδCF-mediated phosphorylation of p73β is associated with accumulation of p73β and induction of p73β-mediated transactivation. By contrast, PKCδCF-induced activation of p73β is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCδCF stimulates p73β-mediated apoptosis and that this response is attenuated with the p73β(S289A) mutant. These findings demonstrate that cleavage of PKCδ to PKCδCF induces apoptosis by a mechanism in part dependent on PKCδCF-mediated phosphorylation of the p73β Ser-289 site. Protein kinase C (PKC) δ is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCδCF) in the apoptotic response of cells to DNA damage. Expression of PKCδCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCδCF associates with p73β, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCδCF phosphorylates the p73β transactivation and DNA-binding domains. One PKCδCF-phosphorylation site has been mapped to Ser-289 in the p73β DNA-binding domain. PKCδCF-mediated phosphorylation of p73β is associated with accumulation of p73β and induction of p73β-mediated transactivation. By contrast, PKCδCF-induced activation of p73β is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCδCF stimulates p73β-mediated apoptosis and that this response is attenuated with the p73β(S289A) mutant. These findings demonstrate that cleavage of PKCδ to PKCδCF induces apoptosis by a mechanism in part dependent on PKCδCF-mediated phosphorylation of the p73β Ser-289 site. protein kinase C catalytic fragment transactivation domain DNA-binding domain ionizing radiation tumor necrosis factor-α oligomerization domain green fluorescent protein glutathione S-transferase DNA-dependent protein kinase The p53 tumor suppressor regulates the transcription of genes involved in control of the cell cycle and apoptosis (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar). Levels of p53 protein increase in the response of cells to DNA damage and certain other forms of stress. Activation of p53-mediated growth arrest or apoptosis prevents the replication of damaged DNA and thereby maintains integrity of the genome (2Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4402) Google Scholar). Two p53 homologs, designated p73 and p63, have been identified that activate transcription from p53-responsive promoters and induce apoptosis (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar, 4Jost C.A. Marin M.C. Kaelin W.G., Jr. Nature. 1997; 389: 191-193Crossref PubMed Scopus (895) Google Scholar, 5Yang A. Kaghad M. Wang Y. Gillett E. Fleming M. Dotsch V. Andrews N. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1821) Google Scholar). Both p73 and p63 share homology with the transactivation, DNA-binding and oligomerization domains of p53. In contrast to p53, p73 and p63 are expressed as multiple isoforms (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar, 5Yang A. Kaghad M. Wang Y. Gillett E. Fleming M. Dotsch V. Andrews N. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1821) Google Scholar). The p73 and p63 isoforms can fold into stable homotetramers through interactions of their oligomerization domains (6Davison T.S. Vagner C. Kaghad M. Ayed A. Caput D. Arrowsmith C.H. J. Biol. Chem. 1999; 274: 18709-18714Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The available findings further indicate that the oligomerization domain of wild-type p53 does not interact with those of p73 or p63 (6Davison T.S. Vagner C. Kaghad M. Ayed A. Caput D. Arrowsmith C.H. J. Biol. Chem. 1999; 274: 18709-18714Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These findings have suggested that p73 and p63 can activate p53-responsive genes by mechanisms independent of p53. Several studies have indicated that p73 is involved in the cellular response to DNA damage. Initial reports showed that, unlike p53, p73 is not subject to accumulation in cells treated with genotoxic agents (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). Other work has shown that the α and β isoforms of p73 interact with the c-Abl tyrosine kinase in the genotoxic stress response. c-Abl is activated by DNA damaging agents and contributes to the induction of apoptosis by p53-dependent and -independent mechanisms (7Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (456) Google Scholar,8Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (177) Google Scholar). The findings demonstrate that c-Abl also stimulates p73-mediated transactivation and that p73 participates in the apoptotic response to DNA damage (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). Moreover, studies have indicated that p73 is transcriptionally regulated by DNA damage and that a binding site in the p73 promoter is activated by p53 and p73 (12Chen X. Zheng Y. Zhu J. Jiang J. Wang J. Oncogene. 2001; 20: 769-774Crossref PubMed Scopus (84) Google Scholar). These findings have provided support for involvement of p73 in response to genotoxic stress. The protein kinase C (PKC)1family of serine/threonine kinases consists of multiple isoforms with conserved catalytic domains (13Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2347) Google Scholar). Differences in their regulatory domains have resulted in classification of the PKC isoforms into conventional, novel, and atypical subgroups. The ubiquitously expressed PKCδ isoform is a member of the novel PKC subgroup and is activated by diacylglycerol or phorbol esters in a calcium-independent manner (14Ono Y. Fujii T. Ogita K. Kikkawa U. Igarahsi K. Nishizuka Y. J. Biol. Chem. 1988; 263: 6927-6932Abstract Full Text PDF PubMed Google Scholar, 15Ogita K. Miyamoto S. Yamaguchi K. Koide H. Fujisawa N. Kikkawa U. Sahara S. Fukami Y. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1592-1596Crossref PubMed Scopus (123) Google Scholar, 16Mizuno K. Kubo K. Saido T. Akita Y. Osada S. Kuroki T. Ohno S. Suzuki K. Eur. J. Biochem. 1991; 202: 931-940Crossref PubMed Scopus (94) Google Scholar). PKCδ is also activated by c-Abl in the cellular response to stress (17Yuan Z.-M. Utsugisawa T. Ishiko T. Nakada S. Huang Y. Kharbanda S. Weichselbaum R. Kufe D. Oncogene. 1998; 16: 1643-1648Crossref PubMed Scopus (126) Google Scholar, 18Sun X., Wu, F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In this regard, treatment of cells with ionizing radiation (IR) is associated with c-Abl-dependent phosphorylation of PKCδ and translocation of PKCδ to the nucleus (17Yuan Z.-M. Utsugisawa T. Ishiko T. Nakada S. Huang Y. Kharbanda S. Weichselbaum R. Kufe D. Oncogene. 1998; 16: 1643-1648Crossref PubMed Scopus (126) Google Scholar). Other studies have demonstrated that PKCδ is activated by caspase-3-mediated cleavage at the third variable region (V3) to a 38-kDa regulatory domain and a 40-kDa constitutively active catalytic fragment (CF) (19Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 20Emoto Y. Kisaki H. Manome Y. Kharbanda S. Kufe D. Blood. 1996; 87: 1990-1996Crossref PubMed Google Scholar). The finding that expression of PKCδCF results in DNA fragmentation has supported a role for PKCδ cleavage in the induction of apoptosis (21Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (446) Google Scholar). The present studies demonstrate that PKCδCF associates with p73β. The results show that PKCδCF phosphorylates p73β in part on Ser-289. The results also demonstrate that PKCδCF-mediated phosphorylation of Ser-289 contributes to p73β-dependent activation and apoptosis. HCT 116-3 (22Boyer J.C. Umar A. Risinger J.I. Lipford J.R. Kane M. Yin S. Barrett J.C. Kolodner R.D. Kunkel T.A. Cancer Res. 1995; 55: 6063-6070PubMed Google Scholar) and 293T cells were grown in Dulbecco’s modified Eagle’s minimum essential medium F-12 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 2 mml-glutamine, and 400 μg/ml geneticin sulfate. SAOS-2 cells and HeLa cells were grown as described earlier (23Endo K. Oki E. Biedermann V. Kojima H. Yoshida K. Johannes F. Kufe D. Datta R. J. Biol. Chem. 2000; 275: 18476-18481Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 24Datta R. Kojima H. Yoshida K. Kufe D. J. Biol. Chem. 1997; 272: 20317-20320Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Cells were treated with 40 μmcisplatin (Sigma), 20 gray IR using a Gammacell 1000 (2.98 gray/min; Atomic Energy of Canada) or 20 ng/ml tumor necrosis factor-α (TNF-α; Promega, Madison, WI) and 10 μg/ml cycloheximide (Sigma). Cell lysates were prepared as described (25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar). Soluble proteins were incubated with anti-p73 (Neomarkers Inc., Fremont, CA), anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-c-Abl (Santa Cruz) for 1 h and precipitated with protein A-Sepharose for an additional 1 h. The resulting immune complexes were washed in lysis buffer, separated by electrophoresis in SDS-PAGE, and transferred to nitrocellulose filters. The residual binding sites were blocked by incubating the filters with 5% dry milk in PBST (phosphate-buffered saline, 0.05% Tween 20) for 1 h at room temperature. Immunoblot analysis was performed with anti-p73, anti-PKCδ, anti-FLAG (Sigma), anti-c-Abl (Calbiochem), or anti-p21 (Oncogene Research Products, Boston, MA). Plasmids expressing glutathione S-transferase (GST)-p73β transactivation domain (TAD; amino acids 1–135), DNA-binding domain (DBD; amino acids 128–313), and oligomerization domain (OD; amino acids 311–499) were prepared by cloning the appropriate PCR product of human p73β into pGEX-2T (Promega). GST-PKCδCF and GST-PKCδCF(K-R) were prepared as described (17Yuan Z.-M. Utsugisawa T. Ishiko T. Nakada S. Huang Y. Kharbanda S. Weichselbaum R. Kufe D. Oncogene. 1998; 16: 1643-1648Crossref PubMed Scopus (126) Google Scholar). Fusion proteins were purified by affinity chromatography using glutathione-Sepharose beads. Plasmids expressing histidine (His)-PKCδCF and His-PKCδCF(K-R) were prepared by cloning PCR products obtained from pKV-PKCδ (21Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (446) Google Scholar) into pET-28α(+) (Novagen, Madison, WI). For fusion protein-binding assays, purified His proteins were incubated with immobilized GST fusion proteins for 1 h at 4 °C. The resulting protein complexes were washed 4 times. The proteins were then separated by SDS-PAGE and subjected to immunoblot analysis with anti-p73 or anti-PKCδ. Gels were also analyzed after staining with Coomassie Blue (Sigma). Purified GST, GST-p73βTAD, GST-p73βDBD, GST-p73βOD, or myelin basic protein (Invitrogen) were incubated in kinase buffer (20 mmTris-HCl, pH 7.4, 20 mm MgCl2, and 4 mm dithiothreitol) containing [γ-32P]ATP or cold ATP. Kinase-active recombinant PKCδFL (Panvera Corp., Madison, WI), His-PKCδCF, or kinase-inactive His-PKCδCF(K-R) was added for 30 min at 30 °C. The reaction products were analyzed by SDS-PAGE and autoradiography. Purified GST-p73βTAD, GST-p73βDBD, and GST-p73βOD was incubated with GST-PKCδCF and [γ-32P]ATP or ATP. The reaction products were subjected to SDS-PAGE. The p73β band was identified by Coomassie Blue staining and excised from the gel. In-gel digestion with trypsin was performed as described (26Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1121) Google Scholar, 27Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1678) Google Scholar). For32P-labeled p73β, the trypsin-digested peptides were fractionated by reverse transcriptase-high performance liquid chromatography. Aliquots of the fractions were assayed for [32P]. Positive fractions were subjected to Edman sequencing. For unlabeled p73β, masses of the trypsin-digested peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectroscopy using a Voyager DE-PRO (Perceptive Biosystem Inc., Framingham, MA). p73β(S289A) was generated using the site-directed mutagenesis kit (Stratagene, La Jolla, CA) to change Ser-289 to Ala. Cells were transfected with FLAG-p73β, GFP-p73β, pKV, pKV-PKCδCF, pKV-PKCδCF(K-R), pGFP-PKCδFL, pGFP-PKCδCF, or pGFP-PKCδCF(K-R) (21Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (446) Google Scholar, 25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar, 28Yuan J. Yankner B.A. Nature. 2000; 407: 802-809Crossref PubMed Scopus (1585) Google Scholar). HeLa cells were transfected by electroporation (Gene Pulsar, Bio-Rad; 0.22 version, 960 μF; efficiency ∼10–20%). 293T cells were transfected in the presence of LipofectAMINE (Invitrogen; efficiency ∼70–80%). SAOS-2 cells were transfected by calcium phosphate (Invitrogen; efficiency ∼15–20%). The cells were harvested at 30–36 h after transfection. Cell lysates were subjected to immunoprecipitation with anti-c-Abl (Santa Cruz Biotechnology) as described (7Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (456) Google Scholar). The immunoprecipitates were incubated in kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 0.1 mm EDTA, 1 mmdithiothreitol, 0.015% Brij 35) containing 5 μCi of [γ-32P]ATP (PerkinElmer Life Sciences, Boston, MA) and 5 μg of GST-Crk-(120–225) or GST-Crk-(120–212) for 20 min at 30 °C. The reaction products were analyzed by SDS-PAGE and autoradiography. SAOS-2 cells were transfected with p21-Luc (29El-Deiry W.S. Tokino T. Waldman T. Oliner J.D. Velculescu V.E. Burrell M. Hill D.E. Healy E. Rees J.L. Hamilton S.R. Cancer Res. 1995; 55: 2910-2919PubMed Google Scholar), β-galactosidase, wild-type p73β, mutant p73β(S289A), PKCδCF, and/or PKCδCF(K-R). Cells were harvested at 36 h after transfection. Luciferase assays were performed as described (Luciferase assay system; Promega). Relative luciferase activity was determined by normalizing luciferase activity with β-galactosidase activity. Analysis of DNA content was performed by staining ethanol-fixed cells with propidium iodide and monitoring by FACScan (BD PharMingen). The number of cells with sub-G1 DNA content were determined with a MODFIT LT program (Verity software house, Topsham, ME). To define proteins that associate with p73, HCT116 cell lysates were subjected to immunoprecipitation with anti-p73. Analysis of the precipitates by SDS-PAGE and staining demonstrated a coprecipitating protein of 78 kDa. Further analysis of the protein by matrix-assisted laser desorption/ionization-mass spectroscopy demonstrated identity with PKCδ (data not shown). To extend these findings, anti-p73 immunoprecipitates from HCT116 cells were subjected to immunoblotting with anti-PKCδ. The results confirmed the association of p73 and full-length PKCδ (PKCδFL) (Fig. 1). PKCδFL is cleaved by caspase-3 to a constitutively active catalytic fragment (PKCδCF) in the apoptotic response of cells to genotoxic stress (19Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 20Emoto Y. Kisaki H. Manome Y. Kharbanda S. Kufe D. Blood. 1996; 87: 1990-1996Crossref PubMed Google Scholar). In concert with these findings, treatment of HCT116 cells with cisplatin was associated with cleavage of PKCδFL to PKCδCF (Fig. 1, second lane). Moreover, analysis of anti-p73 immunoprecipitates from cisplatin-treated HCT116 cells demonstrated coprecipitation of p73 with both PKCδFL and PKCδCF (Fig. 1, third and fourth lanes). To assess regions of p73 involved in the association with PKCδ, GST-p73β fusion proteins (Fig. 2A) containing the TAD (amino acids 1–135), DBD (amino acids 128–313), or OD (amino acids 311–499) were incubated with His-PKCδFL or His-PKCδCF. Immunoblot analysis of the adsorbents with anti-PKCδ demonstrated binding of PKCδFL to each of the three domains (Fig.2B). By contrast, binding of PKCδCF was detectable with p73β TAD and DBD, but not the OD (Fig. 2C). These findings demonstrate that p73β binds to both PKCδFL and PKCδCF. To determine whether p73 is a substrate for PKCδ, the GST-p73β fusion proteins were incubated with PKCδFL and [γ-32P]ATP. Analysis of the reaction products demonstrated a low level of p73β TAD and DBD phosphorylation (Fig. 3A). As a control, PKCδFL-mediated phosphorylation of myelin basic protein was readily detectable (Fig. 3A). In addition, PKCδFL autophosphorylation was detectable in each of the reactions (Fig.3A). Similar studies performed with PKCδCF demonstrated clearly detectable phosphorylation of p73β TAD and DBD, but not OD (Fig. 3B). By contrast, there was no detectable phosphorylation of p73β in reactions containing the kinase-inactive PKCδCF(K-R) mutant (Fig. 3B). To define sites of phosphorylation, p73β was incubated with PKCδCF and [γ-32P]ATP, purified by high performance liquid chromatography, and analyzed by mass spectroscopy. The results showed that p73β is phosphorylated, at least in part, on Ser-289 in the DBD (data not shown). To confirm these findings, Ser-289 was mutated to Ala. Incubation of the p73βDBD(S289A) mutant with PKCδCF showed decreased phosphorylation compared with that obtained with wild-type p73βDBD, but not complete abrogation of the signal (Fig.3C). In concert with these findings, PKCδCF-mediated phosphorylation of p73β(S289A) was decreased compared with that found with wild-type p73β (Fig. 3D). These results demonstrate that PKCδCF phosphorylates the p73β DBD on Ser-289 and that there are additional sites for PKCδCF phosphorylation in the DBD and TAD. To extend the finding that endogenous PKCδFL and PKCδCF associate with p73β in HCT116 cells, we expressed GFP-p73β and PKCδFL or PKCδCF in HeLa cells (Fig. 4A,first to fourth lanes). Immunoblot analysis of anti-GFP immunoprecipitates with anti-PKCδ demonstrated binding of GFP-p73β to endogenous PKCδFL and that the formation of GFP-p73β-PKCδFL complexes is increased by overexpression of PKCδFL (Fig. 4A, fifth to seventh lanes). The results also demonstrate binding of GFP-p73β and PKCδCF (Fig. 4A, eighth lane). Similar results were obtained when FLAG-tagged p73β was expressed with PKCδFL or PKCδCF (data not shown). To determine whether PKCδ affects p73β expression, cells were transfected with FLAG-p73 and GFP-PKCδFL or GFP-PKCδCF. Immunoblot analysis of cell lysates demonstrated that PKCδFL has little if any effect on p73β expression (Fig.4B). By contrast, transfection of PKCδCF was associated with an increase in p73β levels (Fig. 4B). Previous studies have demonstrated that PKCδ activates c-Abl (18Sun X., Wu, F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and that c-Abl interacts with p73 (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). To assess the effects of PKCδCF on c-Abl, cells were transfected with PKCδCF or PKCδCF(K-R). Analysis of anti-c-Abl immunoprecipitates for phosphorylation of Crk-(120–225) demonstrated that expression of PKCδCF, but not PKCδCF(K-R), is associated with c-Abl activation (Fig. 4C). As a control, there was no detectable phosphorylation of Crk-(120–212) which lacks the c-Abl phosphorylation site (Fig. 4C). These findings indicate that PKCδCF-induced activation of c-Abl could function as a second signal in the interaction with p73b. To extend the analysis, HCT116 cells treated with cisplatin were assayed for effects on endogenous p73 expression. The results demonstrate increases in levels of both p73α and p73β (Fig.5A). Moreover, in concert with the finding that PKCδCF and not PKCδFL regulates accumulation of p73, the kinetics of changes in p73 expression corresponded with cleavage of PKCδFL to PKCδCF (Fig. 5B). Similar findings were obtained in irradiated cells (Fig. 5C). IR treatment was associated with cleavage of PKCδFL to PKCδCF, increases in p73β expression, and little if any effect on p73α (Fig.5C). By contrast, there was no increase in p73β expression in cell treatment with TNF-α/cycloheximide (30Johnson B.W. Cepero E. Boise L.H. J. Biol. Chem. 2000; 275: 31546-31553Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) to induce cleavage of PKCδFL by a mechanism independent of DNA damage (Fig. 5D). These findings indicate that p73β is regulated by PKCδCF in the response of cells to DNA damage and not by pro-apoptotic signaling through the TNF-α death receptor. To determine whether PKCδCF affects p73 function, we transfected SAOS2 cells, which are deficient in both p53 (31Diller L. Kassel J. Nelson C. Gryba M. Litwatz G. Gebhardt M. Bressac B. Ozturk M. Baker S. Vogelstein B. Friend S. Mol. Cell. Biol. 1990; 10: 5772-5781Crossref PubMed Scopus (691) Google Scholar) and p73 (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar), with a construct containing the luciferase gene driven by a p53 enhancer from the p21 promoter (p21-Luc) (29El-Deiry W.S. Tokino T. Waldman T. Oliner J.D. Velculescu V.E. Burrell M. Hill D.E. Healy E. Rees J.L. Hamilton S.R. Cancer Res. 1995; 55: 2910-2919PubMed Google Scholar). Co-transfection of p21-Luc with vectors expressing FLAG-p73β and PKCδCF was associated with a 5.1-fold increase in p73 levels as compared with that obtained in the absence of PKCδCF (Fig. 6A). As a control, cotransfection of FLAG-p73β and kinase-inactive PKCδCF(K-R) had no effect on p73β expression (Fig. 6A). To confirm these findings, similar transfection studies were performed with the p73β(S289A) mutant. The results demonstrate that, whereas PKCδCF increases expression of p73β, this response was attenuated with p73β(S289A) (Fig. 6B). In concert with these results, PKCδCF, and not PKCδCF(K-R), stimulated p73β-mediated activation of the luciferase reporter (Fig. 6C). In addition, the effects of PKCδCF were attenuated in part when coexpressed with the p73β(S289A) mutant (Fig. 6C). To further assess the role of PKCδCF in p73β-mediated transactivation, we assayed transfectants for induction of p21. As shown previously (11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar), transfection of p73β was associated with increased expression of p21 protein (Fig.7A). Notably, cotransfection of p73β and PKCδCF, and not PKCδFL or PKCδCF(K-R), induced p21 compared with that in cells transfected with p73β alone (Fig.7A). Analysis at different intervals after transfection demonstrated that induction of p21 corresponds with levels of p73β and PKCδCF expression (Fig. 7B). These results collectively demonstrate that PKCδCF induces p73β-mediated transactivation by a kinase-dependent mechanism. To extend the functional significance of the interaction between PKCδCF and p73β, studies were performed to assess whether PKCδCF affects p73β-induced apoptosis. As shown previously (32Ghayur T. Banerjee S. Hugunin M. Butler D. Herzog L. Carter A. Quintal L. Sekut L. Talanian R. Paskind M. Wong W. Kamen R. Tracey D. Allen H. Nature. 1997; 386: 619-623Crossref PubMed Scopus (1024) Google Scholar), expression of PKCδCF induces an apoptotic response (Fig.8). Notably, coexpression of GFP-p73β and PKCδCF caused a greater increase in the number of apoptotic cells than that achieved collectively with either alone (Fig. 8). Co-transfection of GFP-p73β and PKCδFL was associated with an increase in apoptosis compared with that found with GFP-p73β alone, but not to the extent observed with PKCδCF (Fig. 8). By contrast, cotransfection of GFP-p73β and PKCδ(K-R) had little effect compared with the percentage of apoptotic cells resulting from expression of GFP-p73β alone (Fig. 8). Diverse substrates are subject to caspase-3-mediated cleavage in cells induced to undergo apoptosis. Whereas most substrates of caspase-3 are inactivated, certain proteins, such as PKCδ (19Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 20Emoto Y. Kisaki H. Manome Y. Kharbanda S. Kufe D. Blood. 1996; 87: 1990-1996Crossref PubMed Google Scholar), PKCθ (24Datta R. Kojima H. Yoshida K. Kufe D. J. Biol. Chem. 1997; 272: 20317-20320Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), the p21-activated kinase 2 (33Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (601) Google Scholar), cytosolic phospholipase A2 (34Wissing D. Mouritzen H. Egeblad M. Poirier G.G. Jaattela M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5073-5077Crossref PubMed Scopus (180) Google Scholar), and PITSLRE kinase a2-1 (35Beyaert R. Kidd V.J. Cornelis S. Van de Craen M. Denecker G. Lahti J.M. Gururajan R. Vandenabeele P. Fiers W. J. Biol. Chem. 1997; 272: 11694-11697Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), are activated by caspase-3-mediated proteolysis. Cleavage of PKCδ at a DMQD/N site in the third variable region (V3) generates a 40-kDa fragment that contains the ATP-binding and kinase domains (19Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 20Emoto Y. Kisaki H. Manome Y. Kharbanda S. Kufe D. Blood. 1996; 87: 1990-1996Crossref PubMed Google Scholar). Loss of the N-terminal regulatory sequences results in a catalytic fragment that is constitutively active in the absence of diacylglycerol or phorbol esters (19Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 20Emoto Y. Kisaki H. Manome Y. Kharbanda S. Kufe D. Blood. 1996; 87: 1990-1996Crossref PubMed Google Scholar). The demonstration that overexpression of the PKCδ catalytic fragment (PKCδCF) is associated with chromatin condensation, nuclear fragmentation, appearance of sub-G1 DNA, and lethality has supported a role for PKCδ cleavage in the induction of apoptosis (32Ghayur T. Banerjee S. Hugunin M. Butler D. Herzog L. Carter A. Quintal L. Sekut L. Talanian R. Paskind M. Wong W. Kamen R. Tracey D. Allen H. Nature. 1997; 386: 619-623Crossref PubMed Scopus (1024) Google Scholar). The mechanisms responsible for PKCδCF-induced apoptosis are, however, largely unknown. Certain insights regarding the role of PKCδCF in apoptosis have been derived from the finding that PKCδCF phosphorylates the DNA-dependent protein kinase (DNA-PK) (25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar). Interaction of PKCδCF and DNA-PK inhibits the function of DNA-PK to associate with Ku-DNA complexes and to phosphorylate its downstream target, p53 (25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar). Notably, cells deficient in DNA-PK exhibit partial resistance to apoptosis induced by overexpression of PKCδCF (25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar). These findings have provided support for involvement of PKCδCF in the regulation of an effector of the DNA damage response. The present studies extend the functional role of PKCδCF by demonstrating an interaction with p73. As found previously for DNA-PK (25Bharti A. Kraeft S.-K. Gounder M. Pandey P. Jin S. Yuan Z.-M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (201) Google Scholar), p73 associates constitutively with both PKCδFL and PKCδCF. The significance of the association between p73 and PKCδFL is unclear, but conceivably represents a mechanism in which p73 is regulated by signals that activate PKCδFL in the absence of caspase-3-mediated cleavage. Like other members of the p53 family, the p73α and p73β isoforms contain transactivation DNA-binding and oligomerization domains (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). The two isoforms differ at their C termini as a result of differential splicing of the p73 mRNA (3Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.-C. Valent A. Minty A. Chalon P. Lelias J.-M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). Both isoforms activate p53-responsive promoters and induce apoptosis (4Jost C.A. Marin M.C. Kaelin W.G., Jr. Nature. 1997; 389: 191-193Crossref PubMed Scopus (895) Google Scholar, 36Zhu J. Jiang J. Zhou W. Chen X. Cancer Res. 1998; 58: 5061-5065PubMed Google Scholar). The homology between p53 and p73 suggested that p73 might function in the cellular stress response. Indeed, recent studies showed that p73 is activated by IR- and cisplatin-induced DNA damage and that this response is regulated in part by the c-Abl kinase (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). The findings demonstrate that c-Abl stimulates p73-mediated transactivation (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). Moreover, p73-mediated apoptosis is regulated by a c-Abl-dependent mechanism (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). Other studies have indicated that transcription of the p73 gene is activated by DNA damage (12Chen X. Zheng Y. Zhu J. Jiang J. Wang J. Oncogene. 2001; 20: 769-774Crossref PubMed Scopus (84) Google Scholar). These findings have supported a role for p73 in the genotoxic stress response. The present studies demonstrate that, in addition to c-Abl, p73 is regulated by PKCδ. In this regard, it is noteworthy that c-Abl and PKCδ have been found to interact by cross-activating their kinase functions in the cellular responses to genotoxic and oxidative stress (17Yuan Z.-M. Utsugisawa T. Ishiko T. Nakada S. Huang Y. Kharbanda S. Weichselbaum R. Kufe D. Oncogene. 1998; 16: 1643-1648Crossref PubMed Scopus (126) Google Scholar, 18Sun X., Wu, F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The present results show that both PKCδFL and PKCδCF associate with p73. The results also show that activation by cleavage to PKCδCF is necessary for the detection of p73 phosphorylation. These findings do not exclude the possibility that activation of PKCδ by other mechanisms, such as through interactions with c-Abl, could similarly result in PKCδFL-mediated phosphorylation of p73. Our results further show that PKCδCF phosphorylates p73β, at least in part, on Ser-289 in the DBD. Thus, mutation of Ser-289 to Ala was associated with a decrease in, but not complete abrogation of, p73 phosphorylation. The p73 Ser-289 phosphorylation site (VLGRRSFECRI) is conserved in p53 (LLGRNS269FEVRV) and, based on the p53 structure, is likely to participate in DNA recognition (37Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2123) Google Scholar). These findings indicated that, whereas PKCδCF phosphorylates other sites on p73, Ser-289 phosphorylation can regulate the p73 transactivation function. The functional significance of the interaction between PKCδCF and p73 is supported by the finding that PKCδCF contributes to the accumulation of p73 protein. Cotransfection of PKCδCF, but not PKCδFL, with p73β was associated with an increase in p73β levels. As the generation of endogenous PKCδCF requires a pro-apoptotic signal that activates caspase-3, we treated cells with cisplatin. The results show that cisplatin increases p73α and p73β levels and that the kinetics of the accumulation of these proteins corresponds with cleavage of PKCδFL to PKCδCF. Similar findings were obtained after exposure to IR, but not as a result of TNF-α/cycloheximide-induced cleavage of PKCδFL to PKCδCF. These results indicate that PKCδCF regulates p73 in the response of cells to genotoxic stress and not death receptor signaling. Previous studies have demonstrated that nuclear c-Abl is activated by DNA damaging agents (cisplatin and IR), but not by TNF-α (7Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (456) Google Scholar). Activation of nuclear c-Abl in the response to genotoxic stress is mediated, at least in part, by the protein mutated in ataxia telangiectasia and the DNA-PK (38Kharbanda S. Pandey P. Jin S. Inoue S. Bharti A. Yuan Z.-M. Weichselbaum R. Weaver D. Kufe D. Nature. 1997; 386: 732-735Crossref PubMed Scopus (237) Google Scholar, 39Baskaran R. Wood L.D. Whitaker L.L., Xu, Y. Barlow C. Canman C.E. Morgan S.E. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y.J. Nature. 1997; 387: 516-519Crossref PubMed Scopus (485) Google Scholar, 40Shafman T. Khanna K.K. Kedar P. Spring K. Kozlov S. Yen T. Hobson K. Gatei M. Zhang N. Watters D. Egerton M. Shiloh Y. Kharbanda S. Kufe D. Lavin M.F. Nature. 1997; 387: 520-523Crossref PubMed Scopus (418) Google Scholar). Previous work has also demonstrated that c-Abl contributes to the activation of PKCδ in response of cells to DNA damage (17Yuan Z.-M. Utsugisawa T. Ishiko T. Nakada S. Huang Y. Kharbanda S. Weichselbaum R. Kufe D. Oncogene. 1998; 16: 1643-1648Crossref PubMed Scopus (126) Google Scholar) and that PKCδ activates c-Abl (18Sun X., Wu, F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Importantly, nuclear c-Abl also interacts with p73 and stimulates p73-mediated transactivation (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). These findings and the results of the present study indicate that a second signal involving c-Abl is likely to contribute to PKCδCF-mediated regulation of p73β in the genotoxic stress response. In concert with this TNF-α-induced model, our findings show that, in the absence of nuclear c-Abl activation (7Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (456) Google Scholar), TNF-α-induced generation of PKCδCF is insufficient to result in the induction of p73β. The results obtained by overexpression of PKCδCF suggest that generation of the catalytic fragment is sufficient to increase p73β expression. Thus, overexpression of PKCδCF was associated with induction of p73β-mediated activation of the p21-Luc reporter and p21 gene. Moreover, PKCδCF-mediated accumulation and activation of p73β were attenuated by expression of the p73β(S289A) mutant. The interpretation that PKCδCF is sufficient to activate p73β, however, is contradicted by the finding that TNF-α induces PKCδ cleavage in the absence of p73β activation. This discrepancy can be explained by the observation that overexpression of PKCδCF, but not PKCδCF(K-R), is associated with the activation of nuclear c-Abl, presumably as a result of the nonphysiologically high levels of PKCδCF that are achieved by this approach. These findings and those obtained with genotoxic agents support a model in which p73β activation is in part dependent on PKCδCF-mediated phosphorylation of Ser-289 and that a second signal mediated by c-Abl may be necessary to fully activate p73β. Previous work has shown that p73α and p73β can induce apoptosis (4Jost C.A. Marin M.C. Kaelin W.G., Jr. Nature. 1997; 389: 191-193Crossref PubMed Scopus (895) Google Scholar) and that c-Abl contributes to p73-mediated apoptosis in response to genotoxic stress (9Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (502) Google Scholar, 10Gong J. Costanzo A. Yang H. Melino G. Kaelin W., Jr. Levrero M. Wang J.Y.J. Nature. 1999; 399: 806-809Crossref PubMed Scopus (826) Google Scholar, 11Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y., Lu, H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (538) Google Scholar). Other studies have demonstrated that E2F-1 induces transcription of the p73 gene and that p73 is functional in mediating E2F-1-induced apoptosis (41Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin W.G., Jr. Nature. 2000; 407: 645-648Crossref PubMed Scopus (530) Google Scholar). In concert with these findings and the demonstration that PKCδCF also induces apoptosis (32Ghayur T. Banerjee S. Hugunin M. Butler D. Herzog L. Carter A. Quintal L. Sekut L. Talanian R. Paskind M. Wong W. Kamen R. Tracey D. Allen H. Nature. 1997; 386: 619-623Crossref PubMed Scopus (1024) Google Scholar), the present results demonstrate that the interaction between PKCδCF and p73 contributes to the apoptotic response. As the generation of PKCδCF is conferred by activation of caspase-3, the interaction between PKCδCF and p73 would serve to amplify, rather than initiate, the induction of apoptosis. Thus, cleavage of PKCδFL to the constitutively activated PKCδCF would appear to function as a fail-safe mechanism to ensure that once a cell has committed to undergo apoptosis then pro-apoptotic effectors (i.e. p73) are subject to potentially irreversible induction by PKCδCF-dependent signaling. We are grateful to Kamal Chauhan for excellent technical support." @default.
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• W2014102011 title "p73β Is Regulated by Protein Kinase Cδ Catalytic Fragment Generated in the Apoptotic Response to DNA Damage" @default.
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