- Email: [email protected]
PKCδ stabilizes TAp63 to promote cell apoptosis
FEBS Letters 589 (2015) 2094–2099
journal homepage: www.FEBSLetters.org
PKCd stabilizes TAp63 to promote cell apoptosis Decai Li 1, Chenghua Li 1, Min Wu, Qiongqiong Chen, Qiao Wang, Jian Ren 2, Yujun Zhang ⇑ Center of Growth, Metabolism and Aging, Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, College of Life Sciences, Chengdu 610065, China
a r t i c l e
i n f o
Article history: Received 23 March 2015 Revised 1 June 2015 Accepted 5 June 2015 Available online 23 June 2015 Edited by Zhijie Chang Keywords: PKCd TA isoforms of p63 Apoptosis
a b s t r a c t PKCd and p63 are respectively reported to play important roles in cell apoptosis. But there is no report on interaction between them in regulation of apoptosis. In the present study, we found that PKCd can directly associate and up-regulate TA isoforms of p63 (TAp63) proteins via increasing their stability. PKCd kinase activity and Thr157 site in TAp63 are crucial for this PKCd-induced accumulation of TAp63. PKCd can also enhance TAp63-mediated transcription and cell apoptosis. Taken together, our data indicate that PKCd phosphorylates TAp63 proteins at Thr157 to stabilize them and promote cell apoptosis. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Protein kinase C (PKC) is a family of cytoplasmic lipid-dependent serine/threonine protein kinases in mammals [1]. PKCd is a member of novel PKC isoforms and widely present in various tissues such as brain and epithelial tissue cells. It is reported that PKCd is Ca2+ independent but activated by diacylglycerol, and plays a key role in regulation of cell cycle and apoptosis in many kinds of cells [2]. PKCd can take part in cell apoptosis through physical interaction with other apoptotic regulators. Studies demonstrate that PKCd is phosphorylated and activated by c-Abl under oxidative stress. Activated PKCd in turn can activate c-Abl by phosphorylation which makes mitochondria release cytochrome C and activates caspase-3, consequently promoting cell apoptosis [3]. Other studies have found that PKCd could bind to caspase-3 directly and phosphorylate it to promote cell apoptosis [4]. PKCd also directly phosphorylates tumor suppressor protein p53 at Ser15 and Ser46, resulting in p53 accumulation and cell death [5–7]. In the apoptotic response of cells to DNA damage, PKCd is cleaved into a kinase-active catalytic fragment (PKCdCF), which mediates phosphorylation and accumulation of the p53 homologue p73b [8]. P63 is a new member of the p53 gene family. Due to alternative transcription starting sites, p63 gene encodes two classes of Author contributions: D.L., M.W., Q.C., and Q.W. performed the experiments. C.L., J.R. and Y.Z. devised the hypothesis and designed the experiments. D.L., C.L. and Y.Z. analyzed the data and wrote the manuscript. ⇑ Corresponding author. Fax: +86 28 85415509. E-mail address: [email protected] (Y. Zhang). 1 These authors contribute equally to this work. 2 Deceased.
protein isotypes, TAp63 and DNp63. TAp63 isotypes have a full N-terminal transactivation domain (TAD) each and up-regulate lots of downstream target genes like p53, while DNp63 isoforms only possess incomplete TADs at their N-termini and inhibit transcriptional activity of p53, p73 and TAp63 [9–11]. Furthermore, owing to the C-terminal splicing in different ways, either type of p63 has been subdivided into at least three different subtypes: a, b, c (TAp63a, b, c and DNp63a, b, c) [12]. In recent years, studies have shown that, TAp63 proteins play a key role in multiple apoptosis signaling pathways, and thus participate in apoptosis, despite their very low abundance in somatic cells. Bax, p21, and other downstream target genes can be activated by TAp63, causing cell cycle arrest and apoptosis [12,13]. As TAp63 proteins play important roles in apoptosis, upstream proteins of TAp63 are involved in apoptosis by interacting with them. For example, Plk1 down-regulates TAp63a by phosphorylating it at Ser52, suppressing cell apoptosis [14]; TATA-binding protein-like protein (TLP) and peptidyl-prolyl isomerase Pin1, can promote cell apoptosis by up-regulating TAp63a [15,16]. It has been reported that p53 and p73b are both up-regulated by PKCd, inducing cell apoptosis [6–8]. Whether PKCd has effect on p63 is previously unknown. Here we demonstrate that PKCd stabilizes TAp63 proteins likely via phosphorylating them at Thr157, resulting in enhanced TAp63-mediated transactivation and cell apoptosis. 2. Materials and methods 2.1. Cell culture and drug treatment HeLa, H1299 and HCT116(p53 / ) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) with 10%
http://dx.doi.org/10.1016/j.febslet.2015.06.014 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
2095
heat-inactivated fetal bovine serum (FBS, Hyclone) and 1% penicillin G/streptomycin (Hyclone) at 37 °C in a humidified 5% CO2 incubator. For activation or inhibition of PKCd, 200 nM Phorbol 12-myristate 13-acetate (PMA, Sigma) or 5 lM rottlerin (Sigma) was supplemented 24 h post-transfection and was incubated for 24 more hours.
indicated durations to harvest. Cell lysates were used for immunoblot analysis. Cells were collected at the indicated time points for IB analysis. b-actin was used as a loading control and GFP as a transfection efficiency control.
2.2. Transfection
H1299 cells were transfected with a mixture of Bax-Luc and pRL-TK-Renilla. Cells were harvested at 36 h after transfection and lysed in Passive Lysis Buffer (Promega). Lysates were analyzed for firefly and Renilla luciferase activities using the Dual Luciferase Reagent Assay Kit (Promega).
Cells were transfected with pcDNA-c-Myc-TAp63a, pcDNA-cMyc-TAp63c, pcDNA-c-Myc-DNp63a, pcDNA-c-Myc-DNp63c, pKV-PKCd or pGFP-PKCd plasmid using LipofectAMINE 2000 (Invitrogen). Supplement of 50 ng GFP plasmid pEGFP-N1 was used for each transfection, as a transfection efficiency control. To knockdown PKCd, HeLA cells were transfected with siRNA directed against PKCd or scramble control (Gene Pharma) using LipofectAMINE 2000. After 48 h, cells were harvested for analysis. 2.3. Immunoprecipitation (IP) Cells were lysed in IP lysis buffer [20 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.5% Nonidet P-40, 10 mM KCl, 0.5 m MEDTA, 10% glycerol, 1.5 mM MgCl2]. Cell lysates containing 500 g total protein were pre-cleared with normal mouse IgG (Santa Cruz) and protein G-sepharose (GE health) for 1 h. Then the supernatant was incubated with anti-c-Myc or anti-GFP or normal mouse IgG (Santa Cruz) for 2 h and precipitated with protein G-Sepharose for 2 more hours. The precipitates were washed with lysis buffer three times at 4 °C, resuspended in 30 lL of 2 SDS sample buffer and heated at 100 °C for 10 min, followed by immunoblot (IB) analysis. 2.4. Immunoblot (IB) Cells were collected, washed with phosphate-buffered saline, and resuspended in lysis buffer [20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 1 mM phenylmethylsul-fonyl fluoride (PMSF), 1 lg/mL leupeptin, 1 lg/mL pepstatin, 2 lg/mL aprotinin, 10 mM NaF, 1 mM EDTA, and 1 mM Na3VO4]. Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad). An equal amount of protein was loaded into every well and separated on a 10% SDS– PAGE, then transferred to polyvinylidene difluoride membrane (Bio-Rad), and hybridized to an appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody for subsequent detection with ECL (Millipore). Immunoblot analysis was performed with anti-p63 (Santa Cruz Biotechnology), anti-pho-PKCd (Cell Signaling Technology), anti-PKCd (Santa Cruz Biotechnology), anti-GFP (Santa Cruz Biotechnology), anti-b-actin (Santa Cruz Biotechnology) or anti-PARP (Zenable bioscience). Densitometric analysis was performed with Image Lab software (Bio-Rad). 2.5. Site-directed mutagenesis S147A, T157A, S319A and TS349, 350AA mutant TAp63a plasmids were generated with the site-directed mutagenesis kit (Stratagene), using pcDNA-c-Myc- TAp63a as a template. 2.6. Protein stability assay To test protein stability of TAp63a, HeLa cells were co-transfected with TAp63a and GFP plasmids plus PKCd or empty vector. 24 h post-transfection, cells were supplemented with 50 lg/mL cycloheximide (CHX, Sigma) to block the biosynthesis of nascent proteins. Then cells were cultured for additional
2.7. Luciferase assays
2.8. Flow cytometry analysis (FACS) Cells were fixed in 70% ethanol at 4 °C overnight and stained with 50 lg/mL propidium iodide (Sigma), supplemented with 80 lg/mL RNase A (Sigma) for 40 min at 37 °C in the dark. Then, cells were subjected to FACS analysis with FACScan flow cytometer (Becton Dickson). Data were analyzed using the Cell Quest program.
3. Results 3.1. PKCd can associate and up-regulate TAp63 It has been reported that PKCd can mediate phosphorylation of the tumor suppressors p53 and p73b, increasing their protein levels in cells [5–8]. Since p63 proteins are homologues of p53 and p73b, we wondered if PKCd has similar effects on p63 isoforms. To this end, we co-transfected HeLa cells, which are deficient in p63 proteins, with PKCd and different TA or DN isoforms of p63 plasmids, respectively, using GFP plasmid as a transfection efficiency control. Cell lysates were subjected to immunoblot analysis. The results showed that, TAp63a and TAp63c were up-regulated in the case of overexpression of PKCd, but the two isoforms of DNp63 were not affected (Fig. 1A). This suggests that PKCd can induce an accumulation of TAp63 proteins in cells. To extend these findings, rottlerin, which is a PKCd chemical inhibitor [17], was used to treat the HeLa cells which were transfected with TAp63a plus PKCd or vector control. Our data demonstrate that PKCd-mediated TAp63a up-regulation was abrogated by rottlerin (Fig. 1B). On the other hand, activation of endogenous PKCd with its potent activator, phorbol 12-myristate 13-acetate (PMA) [18], can significantly increase protein level of transfected TAp63a in HeLa cells, while simultaneous supplement of rottlerin can obviously impair this effect (Fig. 1C). Further study demonstrates that siRNA-mediated specific knock-down of endogenous PKCd can also significantly abrogate PMA-induced TAp63a accumulation (Fig. 1D). These data suggest that PKCd up-regulates TAp63 due to its kinase activity. To examine whether PKCd directly interacts with TAp63, HeLa cells were transfected with c-Myc-tagged TAp63a and GFP-tagged PKCd plasmids. The cell lysates were subjected to immunoprecipitation (IP) with anti-C-myc. Immunoblot (IB) analysis demonstrated that the precipitate contains both TAp63a and PKCd (Fig. 1E). To ensure this finding, anti-GFP was used to precipitate PKCd. The results confirmed that TAp63a can be coimmunoprecipitated with PKCd (Fig. 1F). On the other hand, we failed to detect physical interaction between PKCd and DNp63a with co-immunoprecipitation assay (data not shown). In a summary, these results suggest that PKCd physically interacts with TAp63 and induces accumulation of TAp63 proteins in cells due to the kinase activity of PKCd.
2096
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
Fig. 1. PKCd directly associates and up-regulates TAp63. (A) Co-transfection of PKCd increases protein accumulation of TA but not DN isoforms of p63. Different isoforms of p63, including TAp63a, DNp63a, TAp63c, and DNp63c were respectively co-transfected with PKCd (indicated as +) or its vector control (indicated as ) into Hela cells, using GFP as a transfection efficiency control. 24 h post-transfection, cells were harvested and subjected to IB analysis with indicated antibodies. (B) PKCd inhibitor rottlerin abrogates PKCd-induced TAp63a accumulation. HeLa cells transfected with TAp63a and GFP plus PKCd (indicated as +) or its vector control (indicated as ) were treated without (left 2 lanes) or with rottlerin (right 2 lanes). (C) Activation of endogenous PKCd with PMA induces accumulation of TAp63a which can be reversed by rottlerin. HeLa cells transfected with TAp63a and GFP were treated with indicated drugs. (D) Knockdown of endogenous PKCd abrogates PMA-induced accumulation of TAp63a. HeLa cells transfected with TAp63a and GFP were simultaneously transfected with PKCd siRNA or scramble control, or treated with PMA. (E) and (F) PKCd directly associates TAp63a. HeLa cells were co-transfected with c-Myc-TAp63a and GFP-PKCd. Whole-cell lysates were subjected to immunoprecipitation (IP) with normal mouse IgG, monoclonal anti-c-Myc antibody or monoclonal anti-GFP antibody. IP products were subjected to immunoblot (IB) analysis with indicated antibodies.
3.2. PKCd up-regulates TAp63 via increasing the protein stability of TAp63 It is reported that PKCd can phosphorylate p53 and p73 to stabilize them [7,8]. To examine whether PKCd-induced TAp63 accumulation is via enhancing its protein stability, we measured half-life of TAp63a protein in the presence or absence of transfected PKCd. The results showed that the half life of TAp63a was prolonged from about 30 min to more than 120 min by co-transfection of PKCd (Fig. 2A and B). On the other hand, quantitative PCR analysis revealed that mRNA levels of TAp63 were not affected by PKCd (data not shown). These results demonstrate that PKCd can increase the stability of TAp63a protein in the cells, leading to accumulation of TAp63a. 3.3. Thr157 is critical for PKCd-mediated accumulation of TAp63 Since we found that PKCd directly binds to TAp63 and induces the latter’s accumulation through its kinase activity, we wondered which phosphorylation site of TAp63 is critical to this stability regulation. To identify this site, we constructed several point mutations of TAp63a, including S146A, T157A, S319A and TS349, 350AA, to prevent phosphorylation of these putative PKCd modification sites. These four mutants and wild-type TAp63a were respectively co-transfected with PKCd or its vector control in HeLa cells. 24 h post-transfection, cells were harvested and subjected to immunoblot analysis. The results demonstrate that T157A, but not other point mutations, can significantly abrogate PKCd-induced accumulation of TAp63a in cells (Fig. 3A and B). This suggests that phosphorylation of Thr157 mediated by PKCd is crucial to stabilize TAp63. 3.4. PKCd up-regulates TAp63-mediated transactivation and cell apoptosis As homologues of p53, TAp63 proteins function as transcription factors, with lots of target genes, such as p21 and Bax [12,13]. To
investigate whether PKCd-mediated up-regulation of TAp63a can stimulate transcription of its target genes, luciferase reporter assays were performed with H1299 cells, which are deficient in either p63 or p53. Our results demonstrated that TAp63a increased the Bax-luc reporter activity, which was further enhanced by PKCd (Fig. 4A). In addition, PKCd activator PMA also significantly increased transactivity of TAp63a, while PKCd inhibitor rottlerin impaired PKCd-mediated up-regulation of TAp63a transactivity (Fig. 4B). According to our data not shown, TAp63a mediated expression of p21-luc reporter can also be up-regulated by PKCd. These results suggest that PKCd has a positive role in regulating expression of downstream target genes of TAp63a. TAp63a is a well-known tumor suppressor which can induce cell apoptosis via up-regulating its downstream target genes [12]. To determine whether PKCd affects TAp63 function in cell apoptosis, HCT116(p53 / ) cells, which is another cell line deficient in either p63 or p53, were used to overexpress PKCd and TAp63a. 24 h post-transfection, cells were harvested and subjected to IB and FACS analysis. The results demonstrated that TAp63a-induced PARP cleavage, which is a molecular marker of cell apoptosis, was further increased by PKCd overexpression (Fig. 4C). On the other hand, FACS analysis showed that TAp63a increased the percentage of cells in subG1 phase, while simultaneous overexpression of PKCd significantly enhanced this TAp63a-induced subG1 arrest (Fig. 4D). These data suggest that PKCd promotes TAp63a-induced cell apoptosis. 4. Discussion As a Ser/Thr protein kinase, PKCd has been reported to phosphorylate several proteins, including c-Abl, AP21, Caspase-3, p53 and p73b, to promote cell apoptosis [3–8,19]. In this study, we show that TAp63, which are important homologues of p53 and p73b, can be up-regulated by PKCd, resulting in elevated cell apoptosis. We found that PKCd can directly bind to TAp63 proteins and induce their accumulation in cells via increasing their stability; this effect
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
2097
Fig. 3. T157A mutation abrogates PKCd-induced accumulation of TAp63a. (A) HeLa cells were transfected with PKC plasmid or its vector control, together with GFP and wild type (WT) or S146A/T157A/S319A/TS349, 350AA mutant TAp63. 24 h posttransfection, cells were harvested and subjected to IB analysis. (B) Data from 3 independent experiments were quantified and presented as fold activation with standard deviation. The relative level of wild type or each mutant TAp63a in the absence of transfected PKC was respectively set as 1.0. Fig. 2. PKCd increases protein stability of TAp63a. (A) HeLa cells were transfected with TAp63a and GFP plasmids plus PKCd plasmid or its vector control. 24 h posttransfection, the cells were treated with cycloheximide (CHX) for indicated durations followed by IB analysis. Protein levels of TAp63a were normalized with b-actin, and the protein half-life was quantified. The value of 0-min was set as 1.0. (B) The half-life of TAp63a protein was quantified from 3 independent experiments. Data were presented as fold activation with standard deviation.
depends on PKCd kinase activity, and can be abrogated by T157A mutation in TAp63a; PKCd-mediated up-regulation of TAp63 can enhance transcription of its downstream target genes and further induce cell apoptosis. Multiple p63 isoforms are encoded by the p53-related p63 gene. Due to their key roles in a variety of essential biological processes, abundances of p63 proteins are tightly controlled. Protein stability is a major factor affecting protein abundance in cells. Accumulating reports demonstrate that TAp63 proteins are much less stable than DNp63 [16,20]. In response to some extrinsic stress, DNp63 proteins undergo degradation, while TA isoforms of p63 become more stable and accumulate in cells, so as to trigger cell apoptosis [21]. Up to now, it is not well understood how p63 protein stability is regulated. According to our previous study, as well as reports from other groups, phosporylation at multiple sites of p63 proteins can affect their stability. For instance, kinases ATM, CDK2, p70S6K, Plk, p38, HIPK2 and GSK3 were reported to phosphorylate different p63 isoforms and promote their degradation [14,22–25]; c-Abl mediates phosphorylation of Y149, Y171, and Y289 of TAp63 proteins to make them more stable [26], while it also phosphorylates Y55, Y137, and Y308 of DNp63a to destabilize it [27]; the kinase IKKb stabilizes TAp63c but destabilizes DNp63a [28,29]. Recently, we found that phosphorylation of Thr538 in TAp63a as well as the
corresponding site in DNp63a mediated by an unknown kinase may lead to Pin1-mediated isomerization of the adjacent PY motif, consequently preventing them from proteasomal degradation mediated by WWP1 E3 ligase [16]. In the present study, we found that kinase activity of PKCd is essential for its effect on TAp63 protein accumulation. This suggests that PKCd stabilizes TAp63 proteins via phosphorylating them. Compared with DNp63 isoforms, TAp63 proteins possess intact TADs at their N-termini. Our data suggest that PKCd can phosphorylate TAp63 at their Thr157 site to increase their protein stability. Though this site is also included in DNp63 proteins, our data demonstrate that they cannot be stabilized by PKCd (Fig 1A). Further study shows that PKCd can directly bind to TAp63a (Fig. 1E and F), but not DNp63a (data not shown). This indicates that the intact TADs may be necessary for PKCd-mediated phosphorylation of Thr157 in p63 proteins. Further investigations are required to define the underlying mechanism. Endogenous TAp63 proteins are barely detectable in somatic cells in general cases [30]. Ectopic expression or stress-induced accumulation of TAp63 can promote cell cycle arrest and apoptotic cell death through activating p53 pro-apoptotic targets, such as Puma, Bax and p21 [13,26,31–33]. Our data demonstrate that PKCd-induced accumulation of TAp63 may increase transcription of Bax-luc reporter, and enhance cell apoptosis. This conclusion is consistent with the evidence that PKCd can promote cell apoptosis through interaction with other proteins, such as c-Abl, caspase-3, p53 and p73b [3–5,8]. It is previously reported that PKCd promoter contains three p53-like binding sites, and TAp63a can activate transcription of PKCd [34]. So there is likely a
2098
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
Fig. 4. PKCd up-regulates TAp63a-mediated transactivation and cell apoptosis. (A) PKCd up-regulates TAp63a-mediated expression of Bax-Luc reporter. (B) Activation of endogenous PKCd with PMA up-regulates TAp63a-mediated expression of Bax-Luc reporter, while rottlerin impairs PKCd-mediated up-regulation of TAp63a transactivity. H1299 cells were transfected with indicated plasmids and (or) treated with indicated chemicals. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as fold activation with standard deviation (n = 3). (C) and (D) Overexpression of PKCd promotes TAp63-mediated apoptosis. HCT116(p53 / ) cells were transfected with indicated plasmid plus GFP plasmid. 24 h post-transfection, cells were harvested and half of them were subject to IB analysis (C) while another half were used for DNA content-based apoptotic cell analysis by FACS (D). PARP cleavage (C) or subG1 arrest (D) was used as an index of cell apoptosis.
feedforward regulation of PKCd-TAp63 expression in cells: under some circumstances, PKCd is activated and consequently stabilizes TAp63 proteins; upregulated TAp63 can enhance transcription of PKCd, which may further increase TAp63 protein levels to trigger cell apoptosis and cell cycle arrest. In combination with recent evidence supporting PKCd as a tumor suppressor [35], it is possible to develop therapies for some cancer types via activating or restoring PKC activity, using chemical PKC activators such as PMA. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Zhi-Xiong Xiao for numerous stimulating discussions and Han Kang for FACS analysis. This work was supported by National Natural Science Foundation of China (#30870563 to J.R. and #31170729 to Y.Z.). References [1] Mochly-Rosen, D., Das, K. and Grimes, K.V. (2012) Protein kinase C, an elusive therapeutic target? Nat. Rev. Drug Discov. 11, 937–957. [2] Zhao, M., Xia, L. and Chen, G.Q. (2012) Protein kinase cdelta in apoptosis: a brief overview. Arch. Immunol. Ther. Exp. (Warsz) 60, 361–372. [3] Jang, B.C. et al. (2004) Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells. Biochem. Pharmacol. 67, 1819–1829.
[4] Voss, O.H., Kim, S., Wewers, M.D. and Doseff, A.I. (2005) Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3. J. Biol. Chem. 280, 17371–17379. [5] Ryer, E.J., Sakakibara, K., Wang, C., Sarkar, D., Fisher, P.B., Faries, P.L., Kent, K.C. and Liu, B. (2005) Protein kinase C delta induces apoptosis of vascular smooth muscle cells through induction of the tumor suppressor p53 by both p38dependent and p38-independent mechanisms. J. Biol. Chem. 280, 35310– 35317. [6] Abbas, T., White, D., Hui, L., Yoshida, K., Foster, D.A. and Bargonetti, J. (2004) Inhibition of human p53 basal transcription by down-regulation of protein kinase Cdelta. J. Biol. Chem. 279, 9970–9977. [7] Lee, S.J., Kim, D.C., Choi, B.H., Ha, H. and Kim, K.T. (2006) Regulation of p53 by activated protein kinase C-delta during nitric oxide-induced dopaminergic cell death. J. Biol. Chem. 281, 2215–2224. [8] Ren, J., Datta, R., Shioya, H., Li, Y., Oki, E., Biedermann, V., Bharti, A. and Kufe, D. (2002) P73beta is regulated by protein kinase Cdelta catalytic fragment generated in the apoptotic response to DNA damage. J. Biol. Chem. 277, 33758–33765. [9] Yan, W. and Chen, X. (2006) GPX2, a direct target of p63, inhibits oxidative stress-induced apoptosis in a p53-dependent manner. J. Biol. Chem. 281, 7856–7862. [10] Rocco, J.W., Leong, C.O., Kuperwasser, N., DeYoung, M.P. and Ellisen, L.W. (2006) P63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9, 45–56. [11] Guo, X. and Mills, A.A. (2007) P63, cellular senescence and tumor development. Cell Cycle 6, 305–311. [12] Yang, A. et al. (1998) P63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316. [13] Gressner, O. et al. (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24, 2458–2471. [14] Komatsu, S. et al. (2009) Plk1 regulates liver tumor cell death by phosphorylation of TAp63. Oncogene 28, 3631–3641. [15] Suenaga, Y., Ozaki, T., Tanaka, Y., Bu, Y., Kamijo, T., Tokuhisa, T., Nakagawara, A. and Tamura, T.A. (2009) TATA-binding protein (TBP)-like protein is engaged in
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
[16]
[17]
[18] [19] [20]
[21] [22]
[23]
[24]
etoposide-induced apoptosis through transcriptional activation of human TAp63 gene. J. Biol. Chem. 284, 35433–35440. Li, C., Chang, D.L., Yang, Z., Qi, J., Liu, R., He, H., Li, D. and Xiao, Z.X. (2013) Pin1 modulates p63alpha protein stability in regulation of cell survival, proliferation and tumor formation. Cell Death Dis. 4, e943. Gschwendt, M., Muller, H.J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G. and Marks, F. (1994) Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. Niedel, J.E., Kuhn, L.J. and Vandenbark, G.R. (1983) Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 80, 36–40. Nitti, M. et al. (2005) Central role of PKCdelta in glycoxidation-dependent apoptosis of human neurons. Free Radic. Biol. Med. 38, 846–856. Ying, H., Chang, D.L., Zheng, H., McKeon, F. and Xiao, Z.X. (2005) DNA-binding and transactivation activities are essential for TAp63 protein degradation. Mol. Cell. Biol. 25, 6154–6164. Finlan, L.E. and Hupp, T.R. (2007) P63: the phantom of the tumor suppressor. Cell Cycle 6, 1062–1071. Huang, Y., Sen, T., Nagpal, J., Upadhyay, S., Trink, B., Ratovitski, E. and Sidransky, D. (2008) ATM kinase is a master switch for the Delta Np63 alpha phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage. Cell Cycle 7, 2846–2855. Hildesheim, J., Belova, G.I., Tyner, S.D., Zhou, X., Vardanian, L. and Fornace Jr., A.J. (2004) Gadd45a regulates matrix metalloproteinases by suppressing DeltaNp63alpha and beta-catenin via p38 MAP kinase and APC complex activation. Oncogene 23, 1829–1837. Lazzari, C. et al. (2011) HIPK2 phosphorylates DeltaNp63alpha and promotes its degradation in response to DNA damage. Oncogene 30, 4802–4813.
2099
[25] Galli, F. et al. (2010) MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation. J. Cell Sci. 123, 2423–2433. [26] Gonfloni, S. et al. (2009) Inhibition of the c-Abl-TAp63 pathway protects mouse oocytes from chemotherapy-induced death. Nat. Med. 15, 1179–1185. [27] Yuan, M., Luong, P., Hudson, C., Gudmundsdottir, K. and Basu, S. (2010) C-Abl phosphorylation of DeltaNp63alpha is critical for cell viability. Cell Death Dis. 1, e16. [28] MacPartlin, M., Zeng, S.X. and Lu, H. (2008) Phosphorylation and stabilization of TAp63gamma by IkappaB kinase-beta. J. Biol. Chem. 283, 15754–15761. [29] Chatterjee, A., Chang, X., Sen, T., Ravi, R., Bedi, A. and Sidransky, D. (2010) Regulation of p53 family member isoform DeltaNp63alpha by the nuclear factor-kappaB targeting kinase IkappaB kinase beta. Cancer Res. 70, 1419– 1429. [30] Paris, M., Rouleau, M., Puceat, M. and Aberdam, D. (2012) Regulation of skin aging and heart development by TAp63. Cell Death Differ. 19, 186–193. [31] Osada, M. et al. (1998) Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nat. Med. 4, 839–843. [32] Wu, G. et al. (2003) DeltaNp63alpha and TAp63alpha regulate transcription of genes with distinct biological functions in cancer and development. Cancer Res. 63, 2351–2357. [33] Amelio, I., Grespi, F., Annicchiarico-Petruzzelli, M. and Melino, G. (2012) P63 the guardian of human reproduction. Cell Cycle 11, 4545–4551. [34] Ponassi, R., Terrinoni, A., Chikh, A., Rufini, A., Lena, A.M., Sayan, B.S., Melino, G. and Candi, E. (2006) P63 and p73, members of the p53 gene family, transactivate PKCdelta. Biochem. Pharmacol. 72, 1417–1422. [35] Antal, C.E. et al. (2015) Cancer-associated protein kinase C mutations reveal kinase’s role as tumor suppressor. Cell 160, 489–502.
journal homepage: www.FEBSLetters.org
PKCd stabilizes TAp63 to promote cell apoptosis Decai Li 1, Chenghua Li 1, Min Wu, Qiongqiong Chen, Qiao Wang, Jian Ren 2, Yujun Zhang ⇑ Center of Growth, Metabolism and Aging, Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, College of Life Sciences, Chengdu 610065, China
a r t i c l e
i n f o
Article history: Received 23 March 2015 Revised 1 June 2015 Accepted 5 June 2015 Available online 23 June 2015 Edited by Zhijie Chang Keywords: PKCd TA isoforms of p63 Apoptosis
a b s t r a c t PKCd and p63 are respectively reported to play important roles in cell apoptosis. But there is no report on interaction between them in regulation of apoptosis. In the present study, we found that PKCd can directly associate and up-regulate TA isoforms of p63 (TAp63) proteins via increasing their stability. PKCd kinase activity and Thr157 site in TAp63 are crucial for this PKCd-induced accumulation of TAp63. PKCd can also enhance TAp63-mediated transcription and cell apoptosis. Taken together, our data indicate that PKCd phosphorylates TAp63 proteins at Thr157 to stabilize them and promote cell apoptosis. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Protein kinase C (PKC) is a family of cytoplasmic lipid-dependent serine/threonine protein kinases in mammals [1]. PKCd is a member of novel PKC isoforms and widely present in various tissues such as brain and epithelial tissue cells. It is reported that PKCd is Ca2+ independent but activated by diacylglycerol, and plays a key role in regulation of cell cycle and apoptosis in many kinds of cells [2]. PKCd can take part in cell apoptosis through physical interaction with other apoptotic regulators. Studies demonstrate that PKCd is phosphorylated and activated by c-Abl under oxidative stress. Activated PKCd in turn can activate c-Abl by phosphorylation which makes mitochondria release cytochrome C and activates caspase-3, consequently promoting cell apoptosis [3]. Other studies have found that PKCd could bind to caspase-3 directly and phosphorylate it to promote cell apoptosis [4]. PKCd also directly phosphorylates tumor suppressor protein p53 at Ser15 and Ser46, resulting in p53 accumulation and cell death [5–7]. In the apoptotic response of cells to DNA damage, PKCd is cleaved into a kinase-active catalytic fragment (PKCdCF), which mediates phosphorylation and accumulation of the p53 homologue p73b [8]. P63 is a new member of the p53 gene family. Due to alternative transcription starting sites, p63 gene encodes two classes of Author contributions: D.L., M.W., Q.C., and Q.W. performed the experiments. C.L., J.R. and Y.Z. devised the hypothesis and designed the experiments. D.L., C.L. and Y.Z. analyzed the data and wrote the manuscript. ⇑ Corresponding author. Fax: +86 28 85415509. E-mail address: [email protected] (Y. Zhang). 1 These authors contribute equally to this work. 2 Deceased.
protein isotypes, TAp63 and DNp63. TAp63 isotypes have a full N-terminal transactivation domain (TAD) each and up-regulate lots of downstream target genes like p53, while DNp63 isoforms only possess incomplete TADs at their N-termini and inhibit transcriptional activity of p53, p73 and TAp63 [9–11]. Furthermore, owing to the C-terminal splicing in different ways, either type of p63 has been subdivided into at least three different subtypes: a, b, c (TAp63a, b, c and DNp63a, b, c) [12]. In recent years, studies have shown that, TAp63 proteins play a key role in multiple apoptosis signaling pathways, and thus participate in apoptosis, despite their very low abundance in somatic cells. Bax, p21, and other downstream target genes can be activated by TAp63, causing cell cycle arrest and apoptosis [12,13]. As TAp63 proteins play important roles in apoptosis, upstream proteins of TAp63 are involved in apoptosis by interacting with them. For example, Plk1 down-regulates TAp63a by phosphorylating it at Ser52, suppressing cell apoptosis [14]; TATA-binding protein-like protein (TLP) and peptidyl-prolyl isomerase Pin1, can promote cell apoptosis by up-regulating TAp63a [15,16]. It has been reported that p53 and p73b are both up-regulated by PKCd, inducing cell apoptosis [6–8]. Whether PKCd has effect on p63 is previously unknown. Here we demonstrate that PKCd stabilizes TAp63 proteins likely via phosphorylating them at Thr157, resulting in enhanced TAp63-mediated transactivation and cell apoptosis. 2. Materials and methods 2.1. Cell culture and drug treatment HeLa, H1299 and HCT116(p53 / ) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) with 10%
http://dx.doi.org/10.1016/j.febslet.2015.06.014 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
2095
heat-inactivated fetal bovine serum (FBS, Hyclone) and 1% penicillin G/streptomycin (Hyclone) at 37 °C in a humidified 5% CO2 incubator. For activation or inhibition of PKCd, 200 nM Phorbol 12-myristate 13-acetate (PMA, Sigma) or 5 lM rottlerin (Sigma) was supplemented 24 h post-transfection and was incubated for 24 more hours.
indicated durations to harvest. Cell lysates were used for immunoblot analysis. Cells were collected at the indicated time points for IB analysis. b-actin was used as a loading control and GFP as a transfection efficiency control.
2.2. Transfection
H1299 cells were transfected with a mixture of Bax-Luc and pRL-TK-Renilla. Cells were harvested at 36 h after transfection and lysed in Passive Lysis Buffer (Promega). Lysates were analyzed for firefly and Renilla luciferase activities using the Dual Luciferase Reagent Assay Kit (Promega).
Cells were transfected with pcDNA-c-Myc-TAp63a, pcDNA-cMyc-TAp63c, pcDNA-c-Myc-DNp63a, pcDNA-c-Myc-DNp63c, pKV-PKCd or pGFP-PKCd plasmid using LipofectAMINE 2000 (Invitrogen). Supplement of 50 ng GFP plasmid pEGFP-N1 was used for each transfection, as a transfection efficiency control. To knockdown PKCd, HeLA cells were transfected with siRNA directed against PKCd or scramble control (Gene Pharma) using LipofectAMINE 2000. After 48 h, cells were harvested for analysis. 2.3. Immunoprecipitation (IP) Cells were lysed in IP lysis buffer [20 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.5% Nonidet P-40, 10 mM KCl, 0.5 m MEDTA, 10% glycerol, 1.5 mM MgCl2]. Cell lysates containing 500 g total protein were pre-cleared with normal mouse IgG (Santa Cruz) and protein G-sepharose (GE health) for 1 h. Then the supernatant was incubated with anti-c-Myc or anti-GFP or normal mouse IgG (Santa Cruz) for 2 h and precipitated with protein G-Sepharose for 2 more hours. The precipitates were washed with lysis buffer three times at 4 °C, resuspended in 30 lL of 2 SDS sample buffer and heated at 100 °C for 10 min, followed by immunoblot (IB) analysis. 2.4. Immunoblot (IB) Cells were collected, washed with phosphate-buffered saline, and resuspended in lysis buffer [20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 1 mM phenylmethylsul-fonyl fluoride (PMSF), 1 lg/mL leupeptin, 1 lg/mL pepstatin, 2 lg/mL aprotinin, 10 mM NaF, 1 mM EDTA, and 1 mM Na3VO4]. Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad). An equal amount of protein was loaded into every well and separated on a 10% SDS– PAGE, then transferred to polyvinylidene difluoride membrane (Bio-Rad), and hybridized to an appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody for subsequent detection with ECL (Millipore). Immunoblot analysis was performed with anti-p63 (Santa Cruz Biotechnology), anti-pho-PKCd (Cell Signaling Technology), anti-PKCd (Santa Cruz Biotechnology), anti-GFP (Santa Cruz Biotechnology), anti-b-actin (Santa Cruz Biotechnology) or anti-PARP (Zenable bioscience). Densitometric analysis was performed with Image Lab software (Bio-Rad). 2.5. Site-directed mutagenesis S147A, T157A, S319A and TS349, 350AA mutant TAp63a plasmids were generated with the site-directed mutagenesis kit (Stratagene), using pcDNA-c-Myc- TAp63a as a template. 2.6. Protein stability assay To test protein stability of TAp63a, HeLa cells were co-transfected with TAp63a and GFP plasmids plus PKCd or empty vector. 24 h post-transfection, cells were supplemented with 50 lg/mL cycloheximide (CHX, Sigma) to block the biosynthesis of nascent proteins. Then cells were cultured for additional
2.7. Luciferase assays
2.8. Flow cytometry analysis (FACS) Cells were fixed in 70% ethanol at 4 °C overnight and stained with 50 lg/mL propidium iodide (Sigma), supplemented with 80 lg/mL RNase A (Sigma) for 40 min at 37 °C in the dark. Then, cells were subjected to FACS analysis with FACScan flow cytometer (Becton Dickson). Data were analyzed using the Cell Quest program.
3. Results 3.1. PKCd can associate and up-regulate TAp63 It has been reported that PKCd can mediate phosphorylation of the tumor suppressors p53 and p73b, increasing their protein levels in cells [5–8]. Since p63 proteins are homologues of p53 and p73b, we wondered if PKCd has similar effects on p63 isoforms. To this end, we co-transfected HeLa cells, which are deficient in p63 proteins, with PKCd and different TA or DN isoforms of p63 plasmids, respectively, using GFP plasmid as a transfection efficiency control. Cell lysates were subjected to immunoblot analysis. The results showed that, TAp63a and TAp63c were up-regulated in the case of overexpression of PKCd, but the two isoforms of DNp63 were not affected (Fig. 1A). This suggests that PKCd can induce an accumulation of TAp63 proteins in cells. To extend these findings, rottlerin, which is a PKCd chemical inhibitor [17], was used to treat the HeLa cells which were transfected with TAp63a plus PKCd or vector control. Our data demonstrate that PKCd-mediated TAp63a up-regulation was abrogated by rottlerin (Fig. 1B). On the other hand, activation of endogenous PKCd with its potent activator, phorbol 12-myristate 13-acetate (PMA) [18], can significantly increase protein level of transfected TAp63a in HeLa cells, while simultaneous supplement of rottlerin can obviously impair this effect (Fig. 1C). Further study demonstrates that siRNA-mediated specific knock-down of endogenous PKCd can also significantly abrogate PMA-induced TAp63a accumulation (Fig. 1D). These data suggest that PKCd up-regulates TAp63 due to its kinase activity. To examine whether PKCd directly interacts with TAp63, HeLa cells were transfected with c-Myc-tagged TAp63a and GFP-tagged PKCd plasmids. The cell lysates were subjected to immunoprecipitation (IP) with anti-C-myc. Immunoblot (IB) analysis demonstrated that the precipitate contains both TAp63a and PKCd (Fig. 1E). To ensure this finding, anti-GFP was used to precipitate PKCd. The results confirmed that TAp63a can be coimmunoprecipitated with PKCd (Fig. 1F). On the other hand, we failed to detect physical interaction between PKCd and DNp63a with co-immunoprecipitation assay (data not shown). In a summary, these results suggest that PKCd physically interacts with TAp63 and induces accumulation of TAp63 proteins in cells due to the kinase activity of PKCd.
2096
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
Fig. 1. PKCd directly associates and up-regulates TAp63. (A) Co-transfection of PKCd increases protein accumulation of TA but not DN isoforms of p63. Different isoforms of p63, including TAp63a, DNp63a, TAp63c, and DNp63c were respectively co-transfected with PKCd (indicated as +) or its vector control (indicated as ) into Hela cells, using GFP as a transfection efficiency control. 24 h post-transfection, cells were harvested and subjected to IB analysis with indicated antibodies. (B) PKCd inhibitor rottlerin abrogates PKCd-induced TAp63a accumulation. HeLa cells transfected with TAp63a and GFP plus PKCd (indicated as +) or its vector control (indicated as ) were treated without (left 2 lanes) or with rottlerin (right 2 lanes). (C) Activation of endogenous PKCd with PMA induces accumulation of TAp63a which can be reversed by rottlerin. HeLa cells transfected with TAp63a and GFP were treated with indicated drugs. (D) Knockdown of endogenous PKCd abrogates PMA-induced accumulation of TAp63a. HeLa cells transfected with TAp63a and GFP were simultaneously transfected with PKCd siRNA or scramble control, or treated with PMA. (E) and (F) PKCd directly associates TAp63a. HeLa cells were co-transfected with c-Myc-TAp63a and GFP-PKCd. Whole-cell lysates were subjected to immunoprecipitation (IP) with normal mouse IgG, monoclonal anti-c-Myc antibody or monoclonal anti-GFP antibody. IP products were subjected to immunoblot (IB) analysis with indicated antibodies.
3.2. PKCd up-regulates TAp63 via increasing the protein stability of TAp63 It is reported that PKCd can phosphorylate p53 and p73 to stabilize them [7,8]. To examine whether PKCd-induced TAp63 accumulation is via enhancing its protein stability, we measured half-life of TAp63a protein in the presence or absence of transfected PKCd. The results showed that the half life of TAp63a was prolonged from about 30 min to more than 120 min by co-transfection of PKCd (Fig. 2A and B). On the other hand, quantitative PCR analysis revealed that mRNA levels of TAp63 were not affected by PKCd (data not shown). These results demonstrate that PKCd can increase the stability of TAp63a protein in the cells, leading to accumulation of TAp63a. 3.3. Thr157 is critical for PKCd-mediated accumulation of TAp63 Since we found that PKCd directly binds to TAp63 and induces the latter’s accumulation through its kinase activity, we wondered which phosphorylation site of TAp63 is critical to this stability regulation. To identify this site, we constructed several point mutations of TAp63a, including S146A, T157A, S319A and TS349, 350AA, to prevent phosphorylation of these putative PKCd modification sites. These four mutants and wild-type TAp63a were respectively co-transfected with PKCd or its vector control in HeLa cells. 24 h post-transfection, cells were harvested and subjected to immunoblot analysis. The results demonstrate that T157A, but not other point mutations, can significantly abrogate PKCd-induced accumulation of TAp63a in cells (Fig. 3A and B). This suggests that phosphorylation of Thr157 mediated by PKCd is crucial to stabilize TAp63. 3.4. PKCd up-regulates TAp63-mediated transactivation and cell apoptosis As homologues of p53, TAp63 proteins function as transcription factors, with lots of target genes, such as p21 and Bax [12,13]. To
investigate whether PKCd-mediated up-regulation of TAp63a can stimulate transcription of its target genes, luciferase reporter assays were performed with H1299 cells, which are deficient in either p63 or p53. Our results demonstrated that TAp63a increased the Bax-luc reporter activity, which was further enhanced by PKCd (Fig. 4A). In addition, PKCd activator PMA also significantly increased transactivity of TAp63a, while PKCd inhibitor rottlerin impaired PKCd-mediated up-regulation of TAp63a transactivity (Fig. 4B). According to our data not shown, TAp63a mediated expression of p21-luc reporter can also be up-regulated by PKCd. These results suggest that PKCd has a positive role in regulating expression of downstream target genes of TAp63a. TAp63a is a well-known tumor suppressor which can induce cell apoptosis via up-regulating its downstream target genes [12]. To determine whether PKCd affects TAp63 function in cell apoptosis, HCT116(p53 / ) cells, which is another cell line deficient in either p63 or p53, were used to overexpress PKCd and TAp63a. 24 h post-transfection, cells were harvested and subjected to IB and FACS analysis. The results demonstrated that TAp63a-induced PARP cleavage, which is a molecular marker of cell apoptosis, was further increased by PKCd overexpression (Fig. 4C). On the other hand, FACS analysis showed that TAp63a increased the percentage of cells in subG1 phase, while simultaneous overexpression of PKCd significantly enhanced this TAp63a-induced subG1 arrest (Fig. 4D). These data suggest that PKCd promotes TAp63a-induced cell apoptosis. 4. Discussion As a Ser/Thr protein kinase, PKCd has been reported to phosphorylate several proteins, including c-Abl, AP21, Caspase-3, p53 and p73b, to promote cell apoptosis [3–8,19]. In this study, we show that TAp63, which are important homologues of p53 and p73b, can be up-regulated by PKCd, resulting in elevated cell apoptosis. We found that PKCd can directly bind to TAp63 proteins and induce their accumulation in cells via increasing their stability; this effect
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
2097
Fig. 3. T157A mutation abrogates PKCd-induced accumulation of TAp63a. (A) HeLa cells were transfected with PKC plasmid or its vector control, together with GFP and wild type (WT) or S146A/T157A/S319A/TS349, 350AA mutant TAp63. 24 h posttransfection, cells were harvested and subjected to IB analysis. (B) Data from 3 independent experiments were quantified and presented as fold activation with standard deviation. The relative level of wild type or each mutant TAp63a in the absence of transfected PKC was respectively set as 1.0. Fig. 2. PKCd increases protein stability of TAp63a. (A) HeLa cells were transfected with TAp63a and GFP plasmids plus PKCd plasmid or its vector control. 24 h posttransfection, the cells were treated with cycloheximide (CHX) for indicated durations followed by IB analysis. Protein levels of TAp63a were normalized with b-actin, and the protein half-life was quantified. The value of 0-min was set as 1.0. (B) The half-life of TAp63a protein was quantified from 3 independent experiments. Data were presented as fold activation with standard deviation.
depends on PKCd kinase activity, and can be abrogated by T157A mutation in TAp63a; PKCd-mediated up-regulation of TAp63 can enhance transcription of its downstream target genes and further induce cell apoptosis. Multiple p63 isoforms are encoded by the p53-related p63 gene. Due to their key roles in a variety of essential biological processes, abundances of p63 proteins are tightly controlled. Protein stability is a major factor affecting protein abundance in cells. Accumulating reports demonstrate that TAp63 proteins are much less stable than DNp63 [16,20]. In response to some extrinsic stress, DNp63 proteins undergo degradation, while TA isoforms of p63 become more stable and accumulate in cells, so as to trigger cell apoptosis [21]. Up to now, it is not well understood how p63 protein stability is regulated. According to our previous study, as well as reports from other groups, phosporylation at multiple sites of p63 proteins can affect their stability. For instance, kinases ATM, CDK2, p70S6K, Plk, p38, HIPK2 and GSK3 were reported to phosphorylate different p63 isoforms and promote their degradation [14,22–25]; c-Abl mediates phosphorylation of Y149, Y171, and Y289 of TAp63 proteins to make them more stable [26], while it also phosphorylates Y55, Y137, and Y308 of DNp63a to destabilize it [27]; the kinase IKKb stabilizes TAp63c but destabilizes DNp63a [28,29]. Recently, we found that phosphorylation of Thr538 in TAp63a as well as the
corresponding site in DNp63a mediated by an unknown kinase may lead to Pin1-mediated isomerization of the adjacent PY motif, consequently preventing them from proteasomal degradation mediated by WWP1 E3 ligase [16]. In the present study, we found that kinase activity of PKCd is essential for its effect on TAp63 protein accumulation. This suggests that PKCd stabilizes TAp63 proteins via phosphorylating them. Compared with DNp63 isoforms, TAp63 proteins possess intact TADs at their N-termini. Our data suggest that PKCd can phosphorylate TAp63 at their Thr157 site to increase their protein stability. Though this site is also included in DNp63 proteins, our data demonstrate that they cannot be stabilized by PKCd (Fig 1A). Further study shows that PKCd can directly bind to TAp63a (Fig. 1E and F), but not DNp63a (data not shown). This indicates that the intact TADs may be necessary for PKCd-mediated phosphorylation of Thr157 in p63 proteins. Further investigations are required to define the underlying mechanism. Endogenous TAp63 proteins are barely detectable in somatic cells in general cases [30]. Ectopic expression or stress-induced accumulation of TAp63 can promote cell cycle arrest and apoptotic cell death through activating p53 pro-apoptotic targets, such as Puma, Bax and p21 [13,26,31–33]. Our data demonstrate that PKCd-induced accumulation of TAp63 may increase transcription of Bax-luc reporter, and enhance cell apoptosis. This conclusion is consistent with the evidence that PKCd can promote cell apoptosis through interaction with other proteins, such as c-Abl, caspase-3, p53 and p73b [3–5,8]. It is previously reported that PKCd promoter contains three p53-like binding sites, and TAp63a can activate transcription of PKCd [34]. So there is likely a
2098
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
Fig. 4. PKCd up-regulates TAp63a-mediated transactivation and cell apoptosis. (A) PKCd up-regulates TAp63a-mediated expression of Bax-Luc reporter. (B) Activation of endogenous PKCd with PMA up-regulates TAp63a-mediated expression of Bax-Luc reporter, while rottlerin impairs PKCd-mediated up-regulation of TAp63a transactivity. H1299 cells were transfected with indicated plasmids and (or) treated with indicated chemicals. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as fold activation with standard deviation (n = 3). (C) and (D) Overexpression of PKCd promotes TAp63-mediated apoptosis. HCT116(p53 / ) cells were transfected with indicated plasmid plus GFP plasmid. 24 h post-transfection, cells were harvested and half of them were subject to IB analysis (C) while another half were used for DNA content-based apoptotic cell analysis by FACS (D). PARP cleavage (C) or subG1 arrest (D) was used as an index of cell apoptosis.
feedforward regulation of PKCd-TAp63 expression in cells: under some circumstances, PKCd is activated and consequently stabilizes TAp63 proteins; upregulated TAp63 can enhance transcription of PKCd, which may further increase TAp63 protein levels to trigger cell apoptosis and cell cycle arrest. In combination with recent evidence supporting PKCd as a tumor suppressor [35], it is possible to develop therapies for some cancer types via activating or restoring PKC activity, using chemical PKC activators such as PMA. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Zhi-Xiong Xiao for numerous stimulating discussions and Han Kang for FACS analysis. This work was supported by National Natural Science Foundation of China (#30870563 to J.R. and #31170729 to Y.Z.). References [1] Mochly-Rosen, D., Das, K. and Grimes, K.V. (2012) Protein kinase C, an elusive therapeutic target? Nat. Rev. Drug Discov. 11, 937–957. [2] Zhao, M., Xia, L. and Chen, G.Q. (2012) Protein kinase cdelta in apoptosis: a brief overview. Arch. Immunol. Ther. Exp. (Warsz) 60, 361–372. [3] Jang, B.C. et al. (2004) Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells. Biochem. Pharmacol. 67, 1819–1829.
[4] Voss, O.H., Kim, S., Wewers, M.D. and Doseff, A.I. (2005) Regulation of monocyte apoptosis by the protein kinase Cdelta-dependent phosphorylation of caspase-3. J. Biol. Chem. 280, 17371–17379. [5] Ryer, E.J., Sakakibara, K., Wang, C., Sarkar, D., Fisher, P.B., Faries, P.L., Kent, K.C. and Liu, B. (2005) Protein kinase C delta induces apoptosis of vascular smooth muscle cells through induction of the tumor suppressor p53 by both p38dependent and p38-independent mechanisms. J. Biol. Chem. 280, 35310– 35317. [6] Abbas, T., White, D., Hui, L., Yoshida, K., Foster, D.A. and Bargonetti, J. (2004) Inhibition of human p53 basal transcription by down-regulation of protein kinase Cdelta. J. Biol. Chem. 279, 9970–9977. [7] Lee, S.J., Kim, D.C., Choi, B.H., Ha, H. and Kim, K.T. (2006) Regulation of p53 by activated protein kinase C-delta during nitric oxide-induced dopaminergic cell death. J. Biol. Chem. 281, 2215–2224. [8] Ren, J., Datta, R., Shioya, H., Li, Y., Oki, E., Biedermann, V., Bharti, A. and Kufe, D. (2002) P73beta is regulated by protein kinase Cdelta catalytic fragment generated in the apoptotic response to DNA damage. J. Biol. Chem. 277, 33758–33765. [9] Yan, W. and Chen, X. (2006) GPX2, a direct target of p63, inhibits oxidative stress-induced apoptosis in a p53-dependent manner. J. Biol. Chem. 281, 7856–7862. [10] Rocco, J.W., Leong, C.O., Kuperwasser, N., DeYoung, M.P. and Ellisen, L.W. (2006) P63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9, 45–56. [11] Guo, X. and Mills, A.A. (2007) P63, cellular senescence and tumor development. Cell Cycle 6, 305–311. [12] Yang, A. et al. (1998) P63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316. [13] Gressner, O. et al. (2005) TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J. 24, 2458–2471. [14] Komatsu, S. et al. (2009) Plk1 regulates liver tumor cell death by phosphorylation of TAp63. Oncogene 28, 3631–3641. [15] Suenaga, Y., Ozaki, T., Tanaka, Y., Bu, Y., Kamijo, T., Tokuhisa, T., Nakagawara, A. and Tamura, T.A. (2009) TATA-binding protein (TBP)-like protein is engaged in
D. Li et al. / FEBS Letters 589 (2015) 2094–2099
[16]
[17]
[18] [19] [20]
[21] [22]
[23]
[24]
etoposide-induced apoptosis through transcriptional activation of human TAp63 gene. J. Biol. Chem. 284, 35433–35440. Li, C., Chang, D.L., Yang, Z., Qi, J., Liu, R., He, H., Li, D. and Xiao, Z.X. (2013) Pin1 modulates p63alpha protein stability in regulation of cell survival, proliferation and tumor formation. Cell Death Dis. 4, e943. Gschwendt, M., Muller, H.J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G. and Marks, F. (1994) Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. Niedel, J.E., Kuhn, L.J. and Vandenbark, G.R. (1983) Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 80, 36–40. Nitti, M. et al. (2005) Central role of PKCdelta in glycoxidation-dependent apoptosis of human neurons. Free Radic. Biol. Med. 38, 846–856. Ying, H., Chang, D.L., Zheng, H., McKeon, F. and Xiao, Z.X. (2005) DNA-binding and transactivation activities are essential for TAp63 protein degradation. Mol. Cell. Biol. 25, 6154–6164. Finlan, L.E. and Hupp, T.R. (2007) P63: the phantom of the tumor suppressor. Cell Cycle 6, 1062–1071. Huang, Y., Sen, T., Nagpal, J., Upadhyay, S., Trink, B., Ratovitski, E. and Sidransky, D. (2008) ATM kinase is a master switch for the Delta Np63 alpha phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage. Cell Cycle 7, 2846–2855. Hildesheim, J., Belova, G.I., Tyner, S.D., Zhou, X., Vardanian, L. and Fornace Jr., A.J. (2004) Gadd45a regulates matrix metalloproteinases by suppressing DeltaNp63alpha and beta-catenin via p38 MAP kinase and APC complex activation. Oncogene 23, 1829–1837. Lazzari, C. et al. (2011) HIPK2 phosphorylates DeltaNp63alpha and promotes its degradation in response to DNA damage. Oncogene 30, 4802–4813.
2099
[25] Galli, F. et al. (2010) MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation. J. Cell Sci. 123, 2423–2433. [26] Gonfloni, S. et al. (2009) Inhibition of the c-Abl-TAp63 pathway protects mouse oocytes from chemotherapy-induced death. Nat. Med. 15, 1179–1185. [27] Yuan, M., Luong, P., Hudson, C., Gudmundsdottir, K. and Basu, S. (2010) C-Abl phosphorylation of DeltaNp63alpha is critical for cell viability. Cell Death Dis. 1, e16. [28] MacPartlin, M., Zeng, S.X. and Lu, H. (2008) Phosphorylation and stabilization of TAp63gamma by IkappaB kinase-beta. J. Biol. Chem. 283, 15754–15761. [29] Chatterjee, A., Chang, X., Sen, T., Ravi, R., Bedi, A. and Sidransky, D. (2010) Regulation of p53 family member isoform DeltaNp63alpha by the nuclear factor-kappaB targeting kinase IkappaB kinase beta. Cancer Res. 70, 1419– 1429. [30] Paris, M., Rouleau, M., Puceat, M. and Aberdam, D. (2012) Regulation of skin aging and heart development by TAp63. Cell Death Differ. 19, 186–193. [31] Osada, M. et al. (1998) Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nat. Med. 4, 839–843. [32] Wu, G. et al. (2003) DeltaNp63alpha and TAp63alpha regulate transcription of genes with distinct biological functions in cancer and development. Cancer Res. 63, 2351–2357. [33] Amelio, I., Grespi, F., Annicchiarico-Petruzzelli, M. and Melino, G. (2012) P63 the guardian of human reproduction. Cell Cycle 11, 4545–4551. [34] Ponassi, R., Terrinoni, A., Chikh, A., Rufini, A., Lena, A.M., Sayan, B.S., Melino, G. and Candi, E. (2006) P63 and p73, members of the p53 gene family, transactivate PKCdelta. Biochem. Pharmacol. 72, 1417–1422. [35] Antal, C.E. et al. (2015) Cancer-associated protein kinase C mutations reveal kinase’s role as tumor suppressor. Cell 160, 489–502.