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J. Biol. Chem., Vol. 277, Issue 37, 33758-33765, September 13, 2002

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p73 Is Regulated by Protein Kinase C Catalytic Fragment Generated in the Apoptotic Response to DNA Damage^*

Jian Ren, Rakesh Datta^§, Hisashi Shioya, Yongqing Li, Eiji Oki, Verena Biedermann, Ajit Bharti, and Donald Kufe^¶

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, November 6, 2001, and in revised form, June 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC)  is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCCF) in the apoptotic response of cells to DNA damage. Expression of PKCCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCCF associates with p73, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCCF phosphorylates the p73 transactivation and DNA-binding domains. One PKCCF-phosphorylation site has been mapped to Ser-289 in the p73 DNA-binding domain. PKCCF-mediated phosphorylation of p73 is associated with accumulation of p73 and induction of p73-mediated transactivation. By contrast, PKCCF-induced activation of p73 is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCCF stimulates p73-mediated apoptosis and that this response is attenuated with the p73(S289A) mutant. These findings demonstrate that cleavage of PKC to PKCCF induces apoptosis by a mechanism in part dependent on PKCCF-mediated phosphorylation of the p73 Ser-289 site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The p53 tumor suppressor regulates the transcription of genes involved in control of the cell cycle and apoptosis (1). 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 (2). Two p53 homologs, designated p73 and p63, have been identified that activate transcription from p53-responsive promoters and induce apoptosis (3-5). 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 (3, 5). The p73 and p63 isoforms can fold into stable homotetramers through interactions of their oligomerization domains (6). The available findings further indicate that the oligomerization domain of wild-type p53 does not interact with those of p73 or p63 (6). 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 (3). Other work has shown that the  and  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 (7, 8). The findings demonstrate that c-Abl also stimulates p73-mediated transactivation and that p73 participates in the apoptotic response to DNA damage (9-11). 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 (12). These findings have provided support for involvement of p73 in response to genotoxic stress.

The protein kinase C (PKC)¹ family of serine/threonine kinases consists of multiple isoforms with conserved catalytic domains (13). Differences in their regulatory domains have resulted in classification of the PKC isoforms into conventional, novel, and atypical subgroups. The ubiquitously expressed PKC isoform is a member of the novel PKC subgroup and is activated by diacylglycerol or phorbol esters in a calcium-independent manner (14-16). PKC is also activated by c-Abl in the cellular response to stress (17, 18). In this regard, treatment of cells with ionizing radiation (IR) is associated with c-Abl-dependent phosphorylation of PKC and translocation of PKC to the nucleus (17). Other studies have demonstrated that PKC 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) (19, 20). The finding that expression of PKCCF results in DNA fragmentation has supported a role for PKC cleavage in the induction of apoptosis (21).

The present studies demonstrate that PKCCF associates with p73. The results show that PKCCF phosphorylates p73 in part on Ser-289. The results also demonstrate that PKCCF-mediated phosphorylation of Ser-289 contributes to p73-dependent activation and apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- HCT 116-3 (22) 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 mM L-glutamine, and 400 µg/ml geneticin sulfate. SAOS-2 cells and HeLa cells were grown as described earlier (23, 24). Cells were treated with 40 µM cisplatin (Sigma), 20 gray IR using a Gammacell 1000 (2.98 gray/min; Atomic Energy of Canada) or 20 ng/ml tumor necrosis factor- (TNF-; Promega, Madison, WI) and 10 µg/ml cycloheximide (Sigma).

Immunoprecipitation and Immunoblot Analysis-- Cell lysates were prepared as described (25). Soluble proteins were incubated with anti-p73 (Neomarkers Inc., Fremont, CA), anti-PKC (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-PKC, anti-FLAG (Sigma), anti-c-Abl (Calbiochem), or anti-p21 (Oncogene Research Products, Boston, MA).

Fusion Protein-binding Assays-- Plasmids expressing glutathione S-transferase (GST)-p73 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 p73 into pGEX-2T (Promega). GST-PKCCF and GST-PKCCF(K-R) were prepared as described (17). Fusion proteins were purified by affinity chromatography using glutathione-Sepharose beads. Plasmids expressing histidine (His)-PKCCF and His-PKCCF(K-R) were prepared by cloning PCR products obtained from pKV-PKC (21) into pET-28(+) (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-PKC. Gels were also analyzed after staining with Coomassie Blue (Sigma).

In Vitro Phosphorylation Assays-- Purified GST, GST-p73TAD, GST-p73DBD, GST-p73OD, or myelin basic protein (Invitrogen) were incubated in kinase buffer (20 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, and 4 mM dithiothreitol) containing [-³²P]ATP or cold ATP. Kinase-active recombinant PKCFL (Panvera Corp., Madison, WI), His-PKCCF, or kinase-inactive His-PKCCF(K-R) was added for 30 min at 30 °C. The reaction products were analyzed by SDS-PAGE and autoradiography.

Identification of in Vitro Phosphorylation Sites-- Purified GST-p73TAD, GST-p73DBD, and GST-p73OD was incubated with GST-PKCCF and [-³²P]ATP or ATP. The reaction products were subjected to SDS-PAGE. The p73 band was identified by Coomassie Blue staining and excised from the gel. In-gel digestion with trypsin was performed as described (26, 27). For ³²P-labeled p73, 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 p73, 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).

Site-directed Mutagenesis-- p73(S289A) was generated using the site-directed mutagenesis kit (Stratagene, La Jolla, CA) to change Ser-289 to Ala.

Cell Transfections-- Cells were transfected with FLAG-p73, GFP-p73, pKV, pKV-PKCCF, pKV-PKCCF(K-R), pGFP-PKCFL, pGFP-PKCCF, or pGFP-PKCCF(K-R) (21, 25, 28). 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.

Immune Complex Kinase Assays-- Cell lysates were subjected to immunoprecipitation with anti-c-Abl (Santa Cruz Biotechnology) as described (7). The immunoprecipitates were incubated in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 0.015% Brij 35) containing 5 µCi of [-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.

Luciferase Assays-- SAOS-2 cells were transfected with p21-Luc (29), -galactosidase, wild-type p73, mutant p73(S289A), PKCCF, and/or PKCCF(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 -galactosidase activity.

Analysis of Sub-G₁ DNA Content-- 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).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

p73 Associates with PKC in Cells-- 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 PKC (data not shown). To extend these findings, anti-p73 immunoprecipitates from HCT116 cells were subjected to immunoblotting with anti-PKC. The results confirmed the association of p73 and full-length PKC (PKCFL) (Fig. 1). PKCFL is cleaved by caspase-3 to a constitutively active catalytic fragment (PKCCF) in the apoptotic response of cells to genotoxic stress (19, 20). In concert with these findings, treatment of HCT116 cells with cisplatin was associated with cleavage of PKCFL to PKCCF (Fig. 1, second lane). Moreover, analysis of anti-p73 immunoprecipitates from cisplatin-treated HCT116 cells demonstrated coprecipitation of p73 with both PKCFL and PKCCF (Fig. 1, third and fourth lanes).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Association of p73 and PKC in cells. HCT116-3 cells were treated with cisplatinum (CDDP) for 24 h. Lysates were subjected to immunoprecipitation (IP) with anti-p73. Lysates and immunoprecipitates were analyzed by immunoblotting (IB) with anti-PKC. FL, full-length; CF, catalytic fragment.

Binding of p73 and PKC in Vitro-- To assess regions of p73 involved in the association with PKC, GST-p73 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-PKCFL or His-PKCCF. Immunoblot analysis of the adsorbents with anti-PKC demonstrated binding of PKCFL to each of the three domains (Fig. 2B). By contrast, binding of PKCCF was detectable with p73 TAD and DBD, but not the OD (Fig. 2C). These findings demonstrate that p73 binds to both PKCFL and PKCCF.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Binding of p73 and PKC in vitro. A, schematic representation of p73 showing the TAD, DBD, and OD. His-PKCFL (B) or His-PKCCF (C) was incubated with GST, GST-p73TAD, GST-p73 DBD, or GST-p73OD bound to glutathione beads. The adsorbents were subjected to immunoblotting with anti-PKC. Staining the gel with Coomassie Blue demonstrated equal loading of the GST-p73 proteins (data not shown).

PKC Phosphorylates p73-- To determine whether p73 is a substrate for PKC, the GST-p73 fusion proteins were incubated with PKCFL and [-³²P]ATP. Analysis of the reaction products demonstrated a low level of p73 TAD and DBD phosphorylation (Fig. 3A). As a control, PKCFL-mediated phosphorylation of myelin basic protein was readily detectable (Fig. 3A). In addition, PKCFL autophosphorylation was detectable in each of the reactions (Fig. 3A). Similar studies performed with PKCCF demonstrated clearly detectable phosphorylation of p73 TAD and DBD, but not OD (Fig. 3B). By contrast, there was no detectable phosphorylation of p73 in reactions containing the kinase-inactive PKCCF(K-R) mutant (Fig. 3B). To define sites of phosphorylation, p73 was incubated with PKCCF and [-³²P]ATP, purified by high performance liquid chromatography, and analyzed by mass spectroscopy. The results showed that p73 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 p73DBD(S289A) mutant with PKCCF showed decreased phosphorylation compared with that obtained with wild-type p73DBD, but not complete abrogation of the signal (Fig. 3C). In concert with these findings, PKCCF-mediated phosphorylation of p73(S289A) was decreased compared with that found with wild-type p73 (Fig. 3D). These results demonstrate that PKCCF phosphorylates the p73 DBD on Ser-289 and that there are additional sites for PKCCF phosphorylation in the DBD and TAD.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   PKC phosphorylates p73. A, recombinant PKCFL was incubated with GST-p73TAD, GST-p73DBD, GST-p73OD, or myelin basic protein. B, His-PKCCF or His-PKCCF(K-R) was incubated with GST-p73 TAD, GST-p73 DBD, or GST-p73 OD. C, His-PKCCF was incubated with GST-p73 DBD or GST-p73 DBD(S289A). D, His-PKCCF was incubated with GST-p73 or GST-p73(S289A). The kinase assays were initiated by adding [-32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. Input of GST fusion proteins was assessed by staining the gels with Coomassie Blue. The circled P denotes phosphorylation.

PKCCF Regulates p73 Expression in Vivo-- To extend the finding that endogenous PKCFL and PKCCF associate with p73 in HCT116 cells, we expressed GFP-p73 and PKCFL or PKCCF in HeLa cells (Fig. 4A, first to fourth lanes). Immunoblot analysis of anti-GFP immunoprecipitates with anti-PKC demonstrated binding of GFP-p73 to endogenous PKCFL and that the formation of GFP-p73-PKCFL complexes is increased by overexpression of PKCFL (Fig. 4A, fifth to seventh lanes). The results also demonstrate binding of GFP-p73 and PKCCF (Fig. 4A, eighth lane). Similar results were obtained when FLAG-tagged p73 was expressed with PKCFL or PKCCF (data not shown). To determine whether PKC affects p73 expression, cells were transfected with FLAG-p73 and GFP-PKCFL or GFP-PKCCF. Immunoblot analysis of cell lysates demonstrated that PKCFL has little if any effect on p73 expression (Fig. 4B). By contrast, transfection of PKCCF was associated with an increase in p73 levels (Fig. 4B). Previous studies have demonstrated that PKC activates c-Abl (18) and that c-Abl interacts with p73 (9-11). To assess the effects of PKCCF on c-Abl, cells were transfected with PKCCF or PKCCF(K-R). Analysis of anti-c-Abl immunoprecipitates for phosphorylation of Crk-(120-225) demonstrated that expression of PKCCF, but not PKCCF(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 PKCCF-induced activation of c-Abl could function as a second signal in the interaction with p73b.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   PKCCF regulates p73 expression in vivo. A, HeLa cells were transfected with GFP-p73 and pKV-PKCFL or pKV-PKCCF. Lysates were subjected to immunoprecipitation (IP) with anti-GFP and analyzed by immunoblotting with anti-PKC. B, HeLa cells were trans fected with the indicated plasmids. Lysates were analyzed by immunoblotting with anti-FLAG, anti-PKC, or anti-actin. C, 293T cells were transfected with the indicated plasmids. Anti-c-Abl immunoprecipitates were analyzed for phosphorylation of GST-Crk-(120-225) (upper panel) or GST-Crk-(120-212) (second panel). Intensity of the phosphorylation was determined by densitometric scanning and compared with that of the control. Anti-c-Abl immunoprecipitates were also subjected to immunoblotting with anti-c-Abl (third panel). Lysates not subjected to immunoprecipitation were analyzed by immunoblotting with anti-PKC (fourth panel) and anti-actin (lower panel).

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 p73 and p73 (Fig. 5A). Moreover, in concert with the finding that PKCCF and not PKCFL regulates accumulation of p73, the kinetics of changes in p73 expression corresponded with cleavage of PKCFL to PKCCF (Fig. 5B). Similar findings were obtained in irradiated cells (Fig. 5C). IR treatment was associated with cleavage of PKCFL to PKCCF, increases in p73 expression, and little if any effect on p73 (Fig. 5C). By contrast, there was no increase in p73 expression in cell treatment with TNF-/cycloheximide (30) to induce cleavage of PKCFL by a mechanism independent of DNA damage (Fig. 5D). These findings indicate that p73 is regulated by PKCCF in the response of cells to DNA damage and not by pro-apoptotic signaling through the TNF- death receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   PKCCF regulates p73 expression in response of cells to genotoxic stress. HCT116-3 cells were treated with 40 µM cisplatin (CDDP) (A and B), 20 gray IR (C), or 20 ng/ml TNF- and 10 µg/ml cycloheximide (CHX) (D) for the indicated times. Immunoblot analysis of the lysates was performed with anti-p73, anti-PKC, or anti-actin.

PKCCF Regulates p73-mediated Transactivation-- To determine whether PKCCF affects p73 function, we transfected SAOS2 cells, which are deficient in both p53 (31) and p73 (3), with a construct containing the luciferase gene driven by a p53 enhancer from the p21 promoter (p21-Luc) (29). Co-transfection of p21-Luc with vectors expressing FLAG-p73 and PKCCF was associated with a 5.1-fold increase in p73 levels as compared with that obtained in the absence of PKCCF (Fig. 6A). As a control, cotransfection of FLAG-p73 and kinase-inactive PKCCF(K-R) had no effect on p73 expression (Fig. 6A). To confirm these findings, similar transfection studies were performed with the p73(S289A) mutant. The results demonstrate that, whereas PKCCF increases expression of p73, this response was attenuated with p73(S289A) (Fig. 6B). In concert with these results, PKCCF, and not PKCCF(K-R), stimulated p73-mediated activation of the luciferase reporter (Fig. 6C). In addition, the effects of PKCCF were attenuated in part when coexpressed with the p73(S289A) mutant (Fig. 6C).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   PKCCF induces p73 transactivation in vivo. HeLa cells were transfected with p21-Luc, -galactosidase, and the indicated plasmids. A and B, cell lysates prepared from transfected cells were subjected to immunoblot analysis with anti-FLAG or anti-PKC. C, luciferase and -galactosidase assays were performed at 36 h after transfection. Relative luciferase activity was determined by normalizing the luciferase activity with -galactosidase activity. The results are expressed as the mean ± S.D. for two experiments each performed in triplicate.

To further assess the role of PKCCF in p73-mediated transactivation, we assayed transfectants for induction of p21. As shown previously (11), transfection of p73 was associated with increased expression of p21 protein (Fig. 7A). Notably, cotransfection of p73 and PKCCF, and not PKCFL or PKCCF(K-R), induced p21 compared with that in cells transfected with p73 alone (Fig. 7A). Analysis at different intervals after transfection demonstrated that induction of p21 corresponds with levels of p73 and PKCCF expression (Fig. 7B). These results collectively demonstrate that PKCCF induces p73-mediated transactivation by a kinase-dependent mechanism.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   PKCCF regulates the expression of p21. A, HeLa cells were transfected with the indicated p73 and PKC constructs. Cells were harvested at 36 h after transfection. B, HeLa cells were transfected with vectors expressing the indicated constructs. Cells were harvested at the indicated times. Lysates prepared from transfected cells were analyzed by immunoblot analysis with anti-p21 (A and B), anti-FLAG (B), or anti-PKC (B).

PKCCF Regulates p73-mediated Apoptosis-- To extend the functional significance of the interaction between PKCCF and p73, studies were performed to assess whether PKCCF affects p73-induced apoptosis. As shown previously (32), expression of PKCCF induces an apoptotic response (Fig. 8). Notably, coexpression of GFP-p73 and PKCCF caused a greater increase in the number of apoptotic cells than that achieved collectively with either alone (Fig. 8). Co-transfection of GFP-p73 and PKCFL was associated with an increase in apoptosis compared with that found with GFP-p73 alone, but not to the extent observed with PKCCF (Fig. 8). By contrast, cotransfection of GFP-p73 and PKC(K-R) had little effect compared with the percentage of apoptotic cells resulting from expression of GFP-p73 alone (Fig. 8).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   PKCCF regulates p73-mediated apoptosis. HeLa cells were transfected with vectors expressing the indicated plasmids. Cells were assessed for DNA content by flow cytometry at 30 h after transfection.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteolytic Activation of PKC in Apoptotic Cells-- 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 PKC (19, 20), PKC (24), the p21-activated kinase 2 (33), cytosolic phospholipase A2 (34), and PITSLRE kinase a2-1 (35), are activated by caspase-3-mediated proteolysis. Cleavage of PKC at a DMQD/N site in the third variable region (V3) generates a 40-kDa fragment that contains the ATP-binding and kinase domains (19, 20). Loss of the N-terminal regulatory sequences results in a catalytic fragment that is constitutively active in the absence of diacylglycerol or phorbol esters (19, 20). The demonstration that overexpression of the PKC catalytic fragment (PKCCF) is associated with chromatin condensation, nuclear fragmentation, appearance of sub-G₁ DNA, and lethality has supported a role for PKC cleavage in the induction of apoptosis (32). The mechanisms responsible for PKCCF-induced apoptosis are, however, largely unknown.

Certain insights regarding the role of PKCCF in apoptosis have been derived from the finding that PKCCF phosphorylates the DNA-dependent protein kinase (DNA-PK) (25). Interaction of PKCCF and DNA-PK inhibits the function of DNA-PK to associate with Ku-DNA complexes and to phosphorylate its downstream target, p53 (25). Notably, cells deficient in DNA-PK exhibit partial resistance to apoptosis induced by overexpression of PKCCF (25). These findings have provided support for involvement of PKCCF in the regulation of an effector of the DNA damage response. The present studies extend the functional role of PKCCF by demonstrating an interaction with p73. As found previously for DNA-PK (25), p73 associates constitutively with both PKCFL and PKCCF. The significance of the association between p73 and PKCFL is unclear, but conceivably represents a mechanism in which p73 is regulated by signals that activate PKCFL in the absence of caspase-3-mediated cleavage.

Interaction of p73 and PKCCF-- Like other members of the p53 family, the p73 and p73 isoforms contain transactivation DNA-binding and oligomerization domains (3). The two isoforms differ at their C termini as a result of differential splicing of the p73 mRNA (3). Both isoforms activate p53-responsive promoters and induce apoptosis (4, 36). 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 (9-11). The findings demonstrate that c-Abl stimulates p73-mediated transactivation (9-11). Moreover, p73-mediated apoptosis is regulated by a c-Abl-dependent mechanism (9-11). Other studies have indicated that transcription of the p73 gene is activated by DNA damage (12). 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 PKC. In this regard, it is noteworthy that c-Abl and PKC have been found to interact by cross-activating their kinase functions in the cellular responses to genotoxic and oxidative stress (17, 18). The present results show that both PKCFL and PKCCF associate with p73. The results also show that activation by cleavage to PKCCF is necessary for the detection of p73 phosphorylation. These findings do not exclude the possibility that activation of PKC by other mechanisms, such as through interactions with c-Abl, could similarly result in PKCFL-mediated phosphorylation of p73. Our results further show that PKCCF phosphorylates p73, 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 (37). These findings indicated that, whereas PKCCF phosphorylates other sites on p73, Ser-289 phosphorylation can regulate the p73 transactivation function.

Regulation of p73-mediated Transactivation and Apoptosis by PKCCF-- The functional significance of the interaction between PKCCF and p73 is supported by the finding that PKCCF contributes to the accumulation of p73 protein. Cotransfection of PKCCF, but not PKCFL, with p73 was associated with an increase in p73 levels. As the generation of endogenous PKCCF requires a pro-apoptotic signal that activates caspase-3, we treated cells with cisplatin. The results show that cisplatin increases p73 and p73 levels and that the kinetics of the accumulation of these proteins corresponds with cleavage of PKCFL to PKCCF. Similar findings were obtained after exposure to IR, but not as a result of TNF-/cycloheximide-induced cleavage of PKCFL to PKCCF. These results indicate that PKCCF 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- (7). 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 (38-40). Previous work has also demonstrated that c-Abl contributes to the activation of PKC in response of cells to DNA damage (17) and that PKC activates c-Abl (18). Importantly, nuclear c-Abl also interacts with p73 and stimulates p73-mediated transactivation (9-11). These findings and the results of the present study indicate that a second signal involving c-Abl is likely to contribute to PKCCF-mediated regulation of p73 in the genotoxic stress response. In concert with this TNF--induced model, our findings show that, in the absence of nuclear c-Abl activation (7), TNF--induced generation of PKCCF is insufficient to result in the induction of p73.

The results obtained by overexpression of PKCCF suggest that generation of the catalytic fragment is sufficient to increase p73 expression. Thus, overexpression of PKCCF was associated with induction of p73-mediated activation of the p21-Luc reporter and p21 gene. Moreover, PKCCF-mediated accumulation and activation of p73 were attenuated by expression of the p73(S289A) mutant. The interpretation that PKCCF is sufficient to activate p73, however, is contradicted by the finding that TNF- induces PKC cleavage in the absence of p73 activation. This discrepancy can be explained by the observation that overexpression of PKCCF, but not PKCCF(K-R), is associated with the activation of nuclear c-Abl, presumably as a result of the nonphysiologically high levels of PKCCF that are achieved by this approach. These findings and those obtained with genotoxic agents support a model in which p73 activation is in part dependent on PKCCF-mediated phosphorylation of Ser-289 and that a second signal mediated by c-Abl may be necessary to fully activate p73.

Previous work has shown that p73 and p73 can induce apoptosis (4) and that c-Abl contributes to p73-mediated apoptosis in response to genotoxic stress (9-11). Other studies have demonstrated that E2F-1 induces transcription of the p73 gene and that p73 is functional in mediating E2F-1-induced apoptosis (41). In concert with these findings and the demonstration that PKCCF also induces apoptosis (32), the present results demonstrate that the interaction between PKCCF and p73 contributes to the apoptotic response. As the generation of PKCCF is conferred by activation of caspase-3, the interaction between PKCCF and p73 would serve to amplify, rather than initiate, the induction of apoptosis. Thus, cleavage of PKCFL to the constitutively activated PKCCF 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 PKCCF-dependent signaling.

	ACKNOWLEDGEMENT

We are grateful to Kamal Chauhan for excellent technical support.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grants GM58200 and CA55241.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^§ Present address: Biomeasure Inc., Milford, MA 01757.

^¶ To whom correspondence should be addressed.

Published, JBC Papers in Press, July 3, 2002, DOI 10.1074/jbc.M110667200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; CF, catalytic fragment; TAD, transactivation domain; DBD, DNA-binding domain; IR, ionizing radiation; TNF-, tumor necrosis factor-; OD, oligomerization domain; GFP, green fluorescent protein; GST, glutathione S-transferase; DNA-PK, DNA-dependent protein kinase.

	REFERENCES

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