p73 (cid:1) Is Regulated by Protein Kinase C (cid:2) Catalytic Fragment Generated in the Apoptotic Response to DNA Damage*

Protein kinase C (PKC) (cid:2) is cleaved by caspase-3 to a kinase-active catalytic fragment (PKC (cid:2) CF) in the apoptotic response of cells to DNA damage. Expression of PKC (cid:2) CF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKC (cid:2) CF associates with p73 (cid:1) , a structural and functional homologue of the p53 tumor suppressor. The results show that PKC (cid:2) CF phosphorylates the p73 (cid:1) transactivationandDNA-bindingdomains.OnePKC (cid:2) CF-phosphorylation site has been mapped to Ser-289 in the p73 (cid:1) DNA-binding domain. PKC (cid:2) CF-mediated phosphorylation of p73 (cid:1) is associated with accumulation of p73 (cid:1) and induction of p73 (cid:1) -mediated transactivation. By contrast, PKC (cid:2) CF-induced activation of p73 (cid:1) is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKC (cid:2) CF stimulates p73 (cid:1) -mediated apoptosis and that this response is attenuated with the p73 (cid:1)

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 p53mediated 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)(4)(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 PKC␦CF results in DNA fragmentation has supported a role for PKC␦ cleavage in the induction of apoptosis (21).
The present studies demonstrate that PKC␦CF associates with p73␤. The results show that PKC␦CF phosphorylates p73␤ in part on Ser-289. The results also demonstrate that PKC␦CF-mediated phosphorylation of Ser-289 contributes to p73␤-dependent activation and apoptosis.
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).
Identification of in Vitro Phosphorylation Sites-Purified GST-p73␤TAD, GST-p73␤DBD, and GST-p73␤OD was incubated with GST-PKC␦CF and [␥-32 P]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 32 P-labeled p73␤, the trypsin-digested peptides were fractionated by reverse transcriptase-high performance liquid chromatography. Aliquots of the fractions were assayed for [ 32 P]. Positive fractions were subjected to Edman sequencing. For unlabeled p73␤, masses of the trypsin-digested peptides were analyzed by matrixassisted laser desorption/ionization-mass spectroscopy using a Voyager DE-PRO (Perceptive Biosystem Inc., Framingham, MA).
Analysis of Sub-G 1 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-G 1 DNA content were determined with a MODFIT LT program (Verity software house, Topsham, ME).

RESULTS
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 matrixassisted 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␦ (PKC␦FL) (Fig. 1). PKC␦FL is cleaved by caspase-3 to a constitutively active catalytic fragment (PKC␦CF) 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 PKC␦FL to PKC␦CF (Fig. 1, second lane). Moreover, analysis of anti-p73 immunoprecipitates from cisplatin-treated HCT116 cells demonstrated coprecipitation of p73 with both PKC␦FL and PKC␦CF (Fig. 1, third and fourth  lanes).
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-PKC␦FL or His-PKC␦CF. Immunoblot analysis of the adsorbents with anti-PKC␦ demonstrated binding of PKC␦FL to each of the three domains (Fig. 2B). By contrast, binding of PKC␦CF was detectable with p73␤ TAD and DBD, but not the OD (Fig. 2C). These findings demonstrate that p73␤ binds to both PKC␦FL and PKC␦CF.
PKC␦ Phosphorylates p73-To determine whether p73 is a substrate for PKC␦, the GST-p73␤ fusion proteins were incubated with PKC␦FL and [␥-32 P]ATP. Analysis of the reaction products demonstrated a low level of p73␤ TAD and DBD phosphorylation (Fig. 3A). As a control, PKC␦FL-mediated phosphorylation of myelin basic protein was readily detectable (Fig. 3A). In addition, PKC␦FL autophosphorylation was detectable in each of the reactions (Fig. 3A). Similar studies performed with PKC␦CF 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 PKC␦CF(K-R) mutant (Fig. 3B). To define sites of phosphorylation, p73␤ was incubated with PKC␦CF and [␥-32 P]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 p73␤DBD(S289A) mutant with PKC␦CF showed decreased phosphorylation compared with that obtained with wild-type p73␤DBD, but not complete abrogation of the signal (Fig. 3C).

p73␤ Is Regulated by PKC␦
In concert with these findings, PKC␦CF-mediated phosphorylation of p73␤(S289A) was decreased compared with that found with wild-type p73␤ (Fig. 3D). These results demonstrate that PKC␦CF phosphorylates the p73␤ DBD on Ser-289 and that there are additional sites for PKC␦CF phosphorylation in the DBD and TAD.
PKC␦CF Regulates p73 Expression in Vivo-To extend the finding that endogenous PKC␦FL and PKC␦CF associate with p73␤ in HCT116 cells, we expressed GFP-p73␤ and PKC␦FL or PKC␦CF in HeLa cells (Fig. 4A, first to fourth lanes). Immuno-blot analysis of anti-GFP immunoprecipitates with anti-PKC␦ demonstrated binding of GFP-p73␤ to endogenous PKC␦FL and that the formation of GFP-p73␤-PKC␦FL complexes is increased by overexpression of PKC␦FL (Fig. 4A, fifth to seventh lanes). The results also demonstrate binding of GFP-p73␤ and PKC␦CF (Fig. 4A, eighth lane). Similar results were obtained when FLAG-tagged p73␤ was expressed with PKC␦FL or PKC␦CF (data not shown). To determine whether PKC␦ affects p73␤ expression, cells were transfected with FLAG-p73 and GFP-PKC␦FL or GFP-PKC␦CF. Immunoblot analysis of p73␤ Is Regulated by PKC␦ cell lysates demonstrated that PKC␦FL has little if any effect on p73␤ expression (Fig. 4B). By contrast, transfection of PKC␦CF 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 PKC␦CF on c-Abl, cells were transfected with PKC␦CF or PKC␦CF(K-R). Analysis of anti-c-Abl immunoprecipitates for phosphorylation of Crk-(120 -225) demonstrated that expression of PKC␦CF, but not PKC␦CF(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 PKC␦CF-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 p73␣ and p73␤ (Fig. 5A). Moreover, in concert with the finding that PKC␦CF and not PKC␦FL regulates accumulation of p73, the kinetics of changes in p73 expression corresponded with cleavage of PKC␦FL to PKC␦CF (Fig. 5B). Similar findings were obtained in irradiated cells (Fig. 5C). IR treatment was associated with cleavage of PKC␦FL to PKC␦CF, 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 PKC␦FL by a mechanism independent of DNA damage (Fig. 5D). These findings indicate that p73␤ is regulated by PKC␦CF in the response of cells to DNA damage and not by pro-apoptotic signaling through the TNF-␣ death receptor.
PKC␦CF Regulates p73-mediated Transactivation-To determine whether PKC␦CF 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 PKC␦CF was associated with a 5.1-fold increase in p73 levels as compared with that obtained in the absence of PKC␦CF (Fig.  6A). As a control, cotransfection of FLAG-p73␤ and kinaseinactive PKC␦CF(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 PKC␦CF increases expression of p73␤, this response was attenuated with p73␤(S289A) (Fig.  6B). In concert with these results, PKC␦CF, and not PKC␦CF(K-R), stimulated p73␤-mediated activation of the luciferase reporter (Fig. 6C). In addition, the effects of PKC␦CF were attenuated in part when coexpressed with the p73␤(S289A) mutant (Fig. 6C).
To further assess the role of PKC␦CF 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 PKC␦CF, and not PKC␦FL or PKC␦CF(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 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) .   FIG. 4. PKC␦CF regulates p73␤ expression in vivo. A, HeLa cells were transfected with GFP-p73␤ and pKV-PKC␦FL or pKV-PKC␦CF. Lysates were subjected to immunoprecipitation (IP) with anti-GFP and analyzed by immunoblotting with anti-PKC␦. B, HeLa cells were trans-p73␤ Is Regulated by PKC␦ p73␤ and PKC␦CF expression (Fig. 7B). These results collectively demonstrate that PKC␦CF induces p73␤-mediated transactivation by a kinase-dependent mechanism. p73␤ Is Regulated by PKC␦ 33762 PKC␦CF Regulates p73-mediated Apoptosis-To extend the functional significance of the interaction between PKC␦CF and p73␤, studies were performed to assess whether PKC␦CF affects p73␤-induced apoptosis. As shown previously (32), expression of PKC␦CF induces an apoptotic response (Fig. 8). Notably, coexpression of GFP-p73␤ and PKC␦CF 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 PKC␦FL was associated with an increase in apoptosis compared with that found with GFP-p73␤ alone, but not to the extent observed with PKC␦CF (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).

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 (PKC␦CF) is associated with chromatin condensation, nuclear fragmentation, appearance of sub-G 1 DNA, and lethality has supported a role for PKC␦ cleavage in the induction of apoptosis (32). The mechanisms responsible for PKC␦CF-induced apoptosis are, however, largely unknown.
Certain insights regarding the role of PKC␦CF in apoptosis have been derived from the finding that PKC␦CF phosphorylates the DNA-dependent protein kinase (DNA-PK) (25). Interaction of PKC␦CF 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 PKC␦CF (25). These findings have provided support for involvement of PKC␦CF in the regulation of an effector of the DNA damage response. The present studies extend the functional role of PKC␦CF by demonstrating an interaction with p73. As found previously for DNA-PK (25), p73 associates constitutively with both PKC␦FL and PKC␦CF. The significance of the association between p73 and PKC␦FL is unclear, but conceivably represents a mechanism in which p73 is regulated by signals that activate PKC␦FL in the absence of caspase-3-mediated cleavage.
Interaction of p73 and PKC␦CF-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).

p73␤ Is Regulated by PKC␦
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 PKC␦FL and PKC␦CF associate with p73. The results also show that activation by cleavage to PKC␦CF 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 PKC␦FL-mediated phosphorylation of p73. Our results further show that PKC␦CF 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 PKC␦CF phosphorylates other sites on p73, Ser-289 phosphorylation can regulate the p73 transactivation function.
Regulation of p73-mediated Transactivation and Apoptosis by PKC␦CF-The functional significance of the interaction between PKC␦CF and p73 is supported by the finding that PKC␦CF contributes to the accumulation of p73 protein. Cotransfection of PKC␦CF, but not PKC␦FL, with p73␤ was associated with an increase in p73␤ levels. As the generation of endogenous PKC␦CF 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 PKC␦FL to PKC␦CF. Similar findings were obtained after exposure to IR, but not as a result of TNF-␣/ cycloheximide-induced cleavage of PKC␦FL to PKC␦CF. These results indicate that PKC␦CF 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 PKC␦CF-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 PKC␦CF is insufficient to result in the induction of p73␤.
The results obtained by overexpression of PKC␦CF suggest that generation of the catalytic fragment is sufficient to increase p73␤ expression. Thus, overexpression of PKC␦CF was associated with induction of p73␤-mediated activation of the p21-Luc reporter and p21 gene. Moreover, PKC␦CF-mediated accumulation and activation of p73␤ were attenuated by expression of the p73␤(S289A) mutant. The interpretation that PKC␦CF 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 PKC␦CF, but not PKC␦CF(K-R), is associated with the activation of nuclear c-Abl, presumably as a result of the nonphysiologically high levels of PKC␦CF 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 PKC␦CF-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 PKC␦CF also induces apoptosis (32), the present results demonstrate that the interaction between PKC␦CF and p73 contributes to the apoptotic response. As the generation of PKC␦CF is conferred by activation of caspase-3, the interaction between PKC␦CF and p73 would serve to amplify, rather than initiate, the induction of apoptosis. Thus, cleavage of PKC␦FL to the constitutively activated PKC␦CF 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 PKC␦CF-dependent signaling.