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Dephosphorylation of p53 during cell death by N-α-tosyl-l -phenylalanyl chloromethyl ketone
BBRC Biochemical and Biophysical Research Communications 306 (2003) 954–958 www.elsevier.com/locate/ybbrc
Dephosphorylation of p53 during cell death by N-a-tosyl-L -phenylalanyl chloromethyl ketoneq Karam Kim,a Kyung Hee Choi,b Ya-Min Fu,c Gary G. Meadows,c and Cheol O. Joea,* a Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701, Republic of Korea Department of Biology, College of Natural Sciences, Chung-Ang University, Heukseuk Dong, Dongjak Ku, Seoul 156-756, Republic of Korea Department of Pharmaceutical Sciences, Cancer Prevention and Research Center, Washington State University, Pullman, WA 99164-6510, USA
b c
Received 23 May 2003
Abstract The apoptotic function of N-a-tosyl-L -phenylalanyl chloromethyl ketone (TPCK) was investigated in cultured human colorectal carcinoma cells (HCT116). TPCK-induced apoptosis was shown to be p53-dependent in HCT116 cells during the early stage of incubation. The function of p53 was required for TPCK-induced activation of caspase-3 and caspase-7. TPCK promoted dephosphorylation of p53 on serine residues at 6, 9, 46, 376, and 378 in parallel with the activation of p53 transcriptional activity. HCT116 p53)/) cells expressing p53 mutant, in which serine residues at 6, 9, 46, 376, and 378 were replaced by aspartic acids, were resistant to TPCK-induced apoptosis suggesting the requirement of dephosphorylation of p53 on serine residues during TPCKinduced apoptosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: TPCK; p53; Dephosphorylation; Apoptosis; Caspase
N-a-tosyl-L -phenylalanyl chloromethyl ketone (TPCK) was initially described to inhibit chymotrypsin by modifying their active site histone residues. TPCK has been widely used to study signal transduction pathways leading to cell survival and death. Contradictory roles of TPCK on cell death have been presented. TPCK was shown to prevent apoptosis stimulated by Fas [1], taxol [2], daunorubicin [3], and topoisomerase I inhibitor [4] in human T cells. On the other hand, TPCK was reported to promote apoptosis by inhibiting NF-jB activation [5], and c-myc expression [6] in murine B cells. It is likely that TPCK might interfere with the signal transduction pathways that are involved in both cell death and survival. NF-jB, for q Abbreviations: TPCK, N-a-tosyl-L -phenylalanyl chloromethyl ketone; NF-jB, nuclear factor jB; X-gal, 5-bromo-4-chloro-3-indolyl-b-D -galactoside; CHAPS, 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; Ac-DEVD-pNA, N-acetyl–Asp–Glu–Val–Asp p-nitroanilide; PAGE, polyacrylamide gel electrophoresis. * Corresponding author. Fax: +82-42-869-2610. E-mail address: [email protected] (C.O. Joe).
example, prevents apoptosis by inducing the expression of genes that are required for cell survival. The function of NF-jB is also known to be required for the apoptotic pathway in order to express proteins necessary for the progression of apoptosis. Therefore, TPCK, which inhibits NF-jB activation, might induce or prevent apoptosis. Another such molecule that participates in the signal transduction pathways leading to the different cell fate determinations is p53. p53 is a tumor suppressor protein that mediates multiple activities in the regulation of cellular events, including cell cycle arrest, DNA repair, replicative senescence, and apoptosis [7–9]. Compelling evidence suggests that the phosphorylation at serine residues is the key regulatory mechanism that controls the function of p53. More than a dozen phosphorylation sites have been mapped on p53. The relevance of the phosphorylation at specific sites of p53 on its function has also been proposed. The phosphorylation within the N-terminal region of human p53 including serine residues at 6, 9, 15, 20, 33, 37, and 46 [10–15] has been implicated to be associated with p53 transcriptional activation and stabilization after DNA
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01088-X
K. Kim et al. / Biochemical and Biophysical Research Communications 306 (2003) 954–958
damage, while the phosphorylation at serine residues at 315, 378, 389, and 392 [16–19] within the C-terminal region of human p53 was suggested to be linked with the enhanced DNA binding activity of p53. In this study, we explored the relationship between the state of serine phosphorylation of human p53 and the induction of apoptosis in cultured human carcinoma cells after TPCK treatment. Materials and methods Cell lines and reagents. HCT116 p53+/+ and HCT116 p53)/) cells [20] were a generous gift from Deug Y. Shin, Dankook University (South Korea) and were maintained in DMEM (Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS). TPCK was from Calbiochem and antibodies against the specific p53 phosphoserines, caspase-3, caspase-7, and PARP were from Cell Signaling Technology. Antibodies against p21 and p53 (DO-1) were supplied by Santa Cruz. Antibodies against actin and p53 (Ab-1) were purchased from Sigma and Oncogene, respectively. Plasmids and transfection. The full-length human p53 cDNA was amplified by PCR and ligated to pcDNA3-HA digested with EcoRI and XhoI. Mutant p53 5D (S6/9/46/376/378D) was introduced into an EcoRI and XhoI sites of pcDNA3-HA by using QuickChange approach (Stratagene). HCT116 p53)/) cells were co-transfected with pGL2-b-galactosidase construct containing p21 promoter, a control plasmid expressing the luciferase reporter gene (pGL3-luc), and the expression plasmid encoding the full-length wild-type (pcDNA-HAp53) or mutant p53 (pcDNA-HA-p53 5D) using Lipofectamine Plus reagent (Invitrogen). Luciferase activities were determined using Luciferase Assay System (Promega) and used as an internal control to assess the transfection efficiencies. After incubation for 24 h, cells were treated with TPCK and b-galactosidase activity in cell lysate was assayed to evaluate the transcriptional function of p53 using Luminescent b-gal kit (BD Biosciences). Cell death assay and caspase activity assay. TPCK-treated HCT116 p53+/+ and HCT116 p53)/) cells were stained with trypan blue and the number of stained cells were counted using hemocytometer. HCT116 p53)/) cells grown on 35 mm culture dish were transfected with the wild-type p53 or mutant p53 (p53 5D) along with the plasmid encoding b-galactosidase. After incubation for 24 h, 50 lM TPCK was added and transfected cells were fixed and stained with 5-bromo-4chloro-3-indolyl-b-D -galactoside (X-gal). The induction of apoptosis in b-galactosidase positive blue cells expressing p53 derivatives was examined by microscopic observation. The results were expressed as mean values SE from the three independent experiments. HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and cell lysates containing the equal amount of total proteins (15 lg) were mixed with the assay buffer containing 93.7 mM Hepes (pH 7.5), 9.36% sucrose, 0.09% CHAPS, 10 mM DTT, 10 lg/ml leupeptin, and 5 lg/ml pepstatin. Cell lysate proteins were incubated with 200 lM of colorimetric peptide substrate, N-acetyl–Asp–Glu–Val–Asp p-nitroanilide (Ac-DEVD-pNA) for 2 h. The assay was performed at 37 °C and caspase activities were monitored by measuring the absorbance at 405 nm. Dephosphorylation of cellular p53. HCT116 p53+/+ cells (2 106 cells) were washed with PBS and incubated in a phosphate-free DMEM. After incubation for 1 h, cellular proteins were metabolically labeled by adding [32 P]orthophosphate (Amersham Biosciences) to the media (0.2 mCi/ml) and incubated for 4 h. TPCK was then treated for 30 min or 1 h before the lysis. Cell lysate proteins were immunoprecipitated using anti-p53 antibody (DO-1) and phosphorylation of p53 was examined by 10% SDS–PAGE and subsequent autoradiography.
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Results TPCK activates transcriptional and pro-apoptotic function of p53 The data in Fig. 1 demonstrate that TPCK induces p53-dependent cell death. HCT116 p53+/+ cells derived from human colorectal carcinoma underwent cell death after TPCK treatment. However, only the basal level of cell death was observed in TPCK treated HCT116 p53)/) cells under the same condition. The effects of TPCK on the transcriptional activity of p53 were evaluated by the activity of p21 promoter expressing b-galactosidase reporter (Fig. 1B). HCT116 p53)/) cells were transfected with p53 and the activity of p21 promoter was measured after TPCK treatment. The transcriptional activity of p21 promoter sequence linked to b-galactosidase reporter was almost threefold higher in cells incubated for 6 h after 50 lM TPCK treatment than in control cells which were not treated with TPCK (Fig. 1B). Stimulation of p21 expression by p53 trans-
Fig. 1. p53-Dependent induction of cell death by TPCK in HCT116 cells. (A) HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and cell viabilities were measured by trypan blue exclusion method. (B) HCT116 p53)/) cells were co-transfected with pGL2-b-gal containing p21 promoter, a control reporter plasmid (pGL-3-luc), and the expression plasmid encoding the full-length p53 (pcDNA-HA-p53). After incubation for 24 h, 25 or 50 lM TPCK was treated and incubated for additional hours. Cells were harvested and bgalactosidase activities in transfected cells were measured by Luminescent b-gal kit (BD Biosciences). (C) HCT116 p53+/+ cells were treated with 50 lM TPCK for the indicated amount of time. Cell lysates were analyzed by SDS–PAGE followed by immunoblotting using antibodies against actin, p53, and p21.
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K. Kim et al. / Biochemical and Biophysical Research Communications 306 (2003) 954–958
activation was also examined in HCT116 p53+/+ cells after TPCK treatment. Cellular levels of p21 protein were significantly elevated in 3 h after TPCK treatment and maintained thereafter (Fig. 1C). Dephosphorylation of p53 by TPCK The effects of TPCK on the state of phosphorylation of p53 were examined in HCT116 p53+/+ cells (Fig. 2). The cellular levels of p53 in HCT116 p53+/+ cells were not affected by TPCK treatment. However, TPCK-induced dephosphorylation of p53 in HCT116 p53+/+ cells in which cellular proteins including p53 were metabolically labeled with 32 P. Immunoblot analysis using phospho-specific antibodies including P-Ser6, P-Ser9, P-Ser46, and p53 C-terminal specific antibody PAb421, which reacts with non-phosphorylated Ser376 and Ser378, indicates that the phosphorylation at serines 6, 9, 46, 376, and 378 was inhibited in HCT116 p53+/+ cells in response to TPCK treatment. Immunoblot analysis using the corresponding phospho-specific antibodies also revealed that the phosphorylation of p53 at serines 15, 20, 37, and 392 was unaffected by TPCK treatment (data not shown).
Fig. 2. TPCK-induced dephosphorylation of p53. (A) Dephosphorylation of p53 at serine residues by TPCK. TA, transactivation domain; DBD, DNA binding domain; and OD, oligomerization domain. (B) Cellular proteins in HCT116 p53+/+ cells (100 mm) were metabolically labeled with 32 P as described in ÔMaterials and methods.Õ Cellular p53 was immunoprecipitated and the incorporation of 32 P into cellular p53 was examined by 10% SDS–PAGE and autoradiography. (C) HCT116 p53+/+ cells were treated with 50 lM TPCK for the indicated amount of time and phosphorylation of serine residues on p53 in cell lysates proteins were examined by SDS–PAGE followed by immunoblotting using p53 specific phospho-amino acid antibodies. Note that PAb421 reacts with nonphosphorylated serine residues at 376 and 378.
p53-dependent caspase activation by TPCK Since caspases are executor molecules of apoptosis, it was reasonable to hypothesize that p53 might function to activate caspases in order to proceed p53-dependent apoptosis. Data in Fig. 3A implicate an indispensable role of p53 in the induction of apoptosis mediated by TPCK. TPCK treatment evoked caspase activation in HCT116 p53+/+ cells, but not in p53 deficient HCT116 p53)/) cells. Among the six caspases tested, at least caspase-3 and caspase-7 appeared to be activated in HCT116 p53+/+ cells in response to TPCK treatment. The activation of caspase-3 was also evidenced by the cleavage of cellular PARP in TPCK treated HCT116 p53+/+ cells. Both caspase-3 and caspase-7 were not activated by TPCK treatment in HCT116 p53)/) cells. Requirement of p53 dephosphorylation during cell death mediated by TPCK Knowing that TPCK induces dephosphorylation of serine residues on p53, it was necessary to determine whether the activation of transcriptional function of p53 and stimulation of apoptosis in TPCK treated HCT116
Fig. 3. Indispensable role of p53 in caspase activation. (A) HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and caspase activities were measured using colorimetric peptide substrate. (B) The proteolytic cleavages of caspase-3, caspase-7, and PARP were analyzed by immunoblot analysis using antibodies against caspase-3, caspase-7, and PARP, respectively.
K. Kim et al. / Biochemical and Biophysical Research Communications 306 (2003) 954–958
p53+/+ cells were attributable to the dephosphorylation of serine residues on p53. The mutant p53, p53 5D, in which the potential dephosphorylation at serines 6, 9, 46, 376, and 378 was constitutively blocked by substituting serine residues by hydrophilic aspartic acids, was transfected into HCT116 p53)/) cells. Neither the activation of promoter activity of p21 nor the induction of apoptosis was observed in transfected cells expressing p53 5D after TPCK treatment (Fig. 4A). While caspase3 and caspase-7 were immediately activated and cleaved PARP in cells expressing wild-type p53 in response to 50 lM TPCK treatment, cells expressing p53 5D failed to trigger early apoptotic responses including caspase activation and PARP cleavage after TPCK treatment (Fig. 4B). Relatively high amount of cell death observed in p53 5D transfected cells at 9 h postincubation might reflect the cytotoxic potential of TPCK, which is an alkylating agent.
Discussion
Fig. 4. Effects of serine dephosphorylation in p53 on its transcription, TPCK-induced apoptosis, and caspase activation. (A) HCT116 p53)/) cells were transfected with wild-type p53 or mutant p53 5D and the activity of p21 promoter linked to the b-galactosidase reporter was measured after TPCK treatment. For apoptosis assay, HCT116 p53)/) cells were co-transfected with plasmid encoding b-galactosidase with p53 or p53 5D and cell death of transfected cells was measured after staining cells with 5-bromo-4-chloro-3-indolyl-b-D galactoside (X-gal). (B) HCT116 p53)/) cells were transfected with the empty vector (pcDNA3), wild-type p53 or mutant p53 5D, and treated with 50 lM TPCK for the indicated amount of time. Cell lysates were analyzed by immunoblot analysis using antibodies against caspase-3, caspase-7, and PARP.
Acknowledgments
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In this study, we initially report that TPCK (i) dephosphorylates the serine residues both at the C-terminal and N-terminal region of p53, (ii) stimulates the transcriptional activity of p53, and (iii) activates caspases to promote p53-dependent apoptosis in cultured human cells. Our data do not support the general claim that the phosphorylation of p53 increases its transcriptional function. The phosphorylation of p53 has been believed to be linked to the activation of its transcriptional function, protein stability, binding ability with other proteins or DNA, and state of oligomerization [11,13,16,19,21,22]. The biochemical links between the dephosphorylation at serine residues of p53 and the stimulation of apoptosis by TPCK have not been reported. However, there exist some implications that the dephosphorylation might be involved in the regulation of p53 function. Consistent with this speculation, the dephosphorylation of p53 at certain sites in the C-terminal domain after DNA damage has been demonstrated to enhance an association of p53 with 14-3-3 protein which activates p53 function [23,24]. Dephosphorylation of a serine residue in the N-terminal domain of p53 has also been reported in human melanoma A375 cell in response to DNA damage [25]. Our data presented in this study suggest that TPCK-induced apoptosis involves p53 dephosphorylation at serine residues. There are unsolved questions to be determined regarding this hypothesis. The roles of the dephosphorylation of serine residues in p53 in the signal transduction leading to the pro-apoptotic function demand further investigation. The involvement of dephosphorylation in TPCK-mediated apoptosis does not seem to be a restricted phenomenon that occurs in HCT116 cells derived from human colorectal carcinoma, since the dephosphorylation of p53 on serine residues was also observed in TPCK treated Cos cells derived from monkey kidney (data not shown). Our findings might provide some additional insights into the mechanisms whereby the state of p53 phosphorylation is modulated by TPCK to regulate signaling pathways influencing cell death and survival.
We thank Dr. John Blenis and Dr. Bryan Ballif for important communications and critical reading of the manuscript. This work was supported by the Grant 2002-CP0432 from Korea Research Foundation, South Korea.
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[14] R.S. Tibbetts, K.M. Brumbaugh, J.M. Williams, J.N. Sarkaria, W.A. Cliby, S.Y. Shieh, Y. Taya, C. Prives, R.T. Abraham, A role for ATR in the DNA damage-induced phosphorylation of p53, Genes Dev. 13 (1999) 152–157. [15] K. Oda, H. Arakawa, T. Tanaka, K. Matsuda, C. Tanikawa, T. Mori, H. Nishimori, K. Tamai, T. Tokino, Y. Nakamura, Y. Taya, p53AIP1, a potential mediator of p53-dependent apoptosis, its regulation by Ser-46-phosphorylated p53, Cell 102 (2000) 849– 862. [16] C. Huang, W.Y. Ma, A. Maxiner, Y. Sun, Z. Dong, p38 kinase mediates UV-induced phosphorylation of p53 protein at serine 389, J. Biol. Chem. 274 (1999) 12229–12235. [17] T.R. Hupp, D.W. Meek, C.A. Midgley, D.P. Lane, Regulation of the specific DNA binding function of p53, Cell 71 (1992) 875–886. [18] H. Lu, R.P. Fisher, P. Bailey, A.J. Levine, The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53 enhancing its sequence-specific DNA binding activity in vitro, Mol. Cell Biol. 17 (1997) 5923–5934. [19] I. Takenaka, F. Morin, B.R. Seizinger, N. Kley, Regulation of the sequence-specific DNA binding function of p53 by protein kinase C and protein phosphatases, J. Biol. Chem. 270 (1995) 5405–5411. [20] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, J.M. Sedivy, K.W. Kinzler, B. Vogelstein, Requirement for p53 and p21 to sustain G2 arrest after DNA damage, Science 282 (1998) 1497–1501. [21] S.Y. Shieh, Y. Taya, C. Prives, DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization, EMBO J. 18 (1999) 1815–1823. [22] K. Sakaguchi, H. Sakamoto, M.S. Lewis, C.W. Anderson, J.W. Erickson, E. Appella, D. Xie, Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53, Biochemistry 36 (1997) 10117–10124. [23] M.J. Waterman, E.S. Stavridi, J.L. Waterman, T.D. Halazonetis, ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins, Nat. Genet. 19 (1998) 175–178. [24] E.S. Stavridi, N.H. Chehab, A. Malikzay, T.D. Halazonetis, Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest, Cancer Res. 61 (2001) 7030–7033. [25] A.L. Craig, J.P. Blaydes, L.R. Burch, A.M. Thompson, T.R. Hupp, Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation, Oncogene 18 (1999) 6305– 6312.
Dephosphorylation of p53 during cell death by N-a-tosyl-L -phenylalanyl chloromethyl ketoneq Karam Kim,a Kyung Hee Choi,b Ya-Min Fu,c Gary G. Meadows,c and Cheol O. Joea,* a Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701, Republic of Korea Department of Biology, College of Natural Sciences, Chung-Ang University, Heukseuk Dong, Dongjak Ku, Seoul 156-756, Republic of Korea Department of Pharmaceutical Sciences, Cancer Prevention and Research Center, Washington State University, Pullman, WA 99164-6510, USA
b c
Received 23 May 2003
Abstract The apoptotic function of N-a-tosyl-L -phenylalanyl chloromethyl ketone (TPCK) was investigated in cultured human colorectal carcinoma cells (HCT116). TPCK-induced apoptosis was shown to be p53-dependent in HCT116 cells during the early stage of incubation. The function of p53 was required for TPCK-induced activation of caspase-3 and caspase-7. TPCK promoted dephosphorylation of p53 on serine residues at 6, 9, 46, 376, and 378 in parallel with the activation of p53 transcriptional activity. HCT116 p53)/) cells expressing p53 mutant, in which serine residues at 6, 9, 46, 376, and 378 were replaced by aspartic acids, were resistant to TPCK-induced apoptosis suggesting the requirement of dephosphorylation of p53 on serine residues during TPCKinduced apoptosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: TPCK; p53; Dephosphorylation; Apoptosis; Caspase
N-a-tosyl-L -phenylalanyl chloromethyl ketone (TPCK) was initially described to inhibit chymotrypsin by modifying their active site histone residues. TPCK has been widely used to study signal transduction pathways leading to cell survival and death. Contradictory roles of TPCK on cell death have been presented. TPCK was shown to prevent apoptosis stimulated by Fas [1], taxol [2], daunorubicin [3], and topoisomerase I inhibitor [4] in human T cells. On the other hand, TPCK was reported to promote apoptosis by inhibiting NF-jB activation [5], and c-myc expression [6] in murine B cells. It is likely that TPCK might interfere with the signal transduction pathways that are involved in both cell death and survival. NF-jB, for q Abbreviations: TPCK, N-a-tosyl-L -phenylalanyl chloromethyl ketone; NF-jB, nuclear factor jB; X-gal, 5-bromo-4-chloro-3-indolyl-b-D -galactoside; CHAPS, 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; Ac-DEVD-pNA, N-acetyl–Asp–Glu–Val–Asp p-nitroanilide; PAGE, polyacrylamide gel electrophoresis. * Corresponding author. Fax: +82-42-869-2610. E-mail address: [email protected] (C.O. Joe).
example, prevents apoptosis by inducing the expression of genes that are required for cell survival. The function of NF-jB is also known to be required for the apoptotic pathway in order to express proteins necessary for the progression of apoptosis. Therefore, TPCK, which inhibits NF-jB activation, might induce or prevent apoptosis. Another such molecule that participates in the signal transduction pathways leading to the different cell fate determinations is p53. p53 is a tumor suppressor protein that mediates multiple activities in the regulation of cellular events, including cell cycle arrest, DNA repair, replicative senescence, and apoptosis [7–9]. Compelling evidence suggests that the phosphorylation at serine residues is the key regulatory mechanism that controls the function of p53. More than a dozen phosphorylation sites have been mapped on p53. The relevance of the phosphorylation at specific sites of p53 on its function has also been proposed. The phosphorylation within the N-terminal region of human p53 including serine residues at 6, 9, 15, 20, 33, 37, and 46 [10–15] has been implicated to be associated with p53 transcriptional activation and stabilization after DNA
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01088-X
K. Kim et al. / Biochemical and Biophysical Research Communications 306 (2003) 954–958
damage, while the phosphorylation at serine residues at 315, 378, 389, and 392 [16–19] within the C-terminal region of human p53 was suggested to be linked with the enhanced DNA binding activity of p53. In this study, we explored the relationship between the state of serine phosphorylation of human p53 and the induction of apoptosis in cultured human carcinoma cells after TPCK treatment. Materials and methods Cell lines and reagents. HCT116 p53+/+ and HCT116 p53)/) cells [20] were a generous gift from Deug Y. Shin, Dankook University (South Korea) and were maintained in DMEM (Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS). TPCK was from Calbiochem and antibodies against the specific p53 phosphoserines, caspase-3, caspase-7, and PARP were from Cell Signaling Technology. Antibodies against p21 and p53 (DO-1) were supplied by Santa Cruz. Antibodies against actin and p53 (Ab-1) were purchased from Sigma and Oncogene, respectively. Plasmids and transfection. The full-length human p53 cDNA was amplified by PCR and ligated to pcDNA3-HA digested with EcoRI and XhoI. Mutant p53 5D (S6/9/46/376/378D) was introduced into an EcoRI and XhoI sites of pcDNA3-HA by using QuickChange approach (Stratagene). HCT116 p53)/) cells were co-transfected with pGL2-b-galactosidase construct containing p21 promoter, a control plasmid expressing the luciferase reporter gene (pGL3-luc), and the expression plasmid encoding the full-length wild-type (pcDNA-HAp53) or mutant p53 (pcDNA-HA-p53 5D) using Lipofectamine Plus reagent (Invitrogen). Luciferase activities were determined using Luciferase Assay System (Promega) and used as an internal control to assess the transfection efficiencies. After incubation for 24 h, cells were treated with TPCK and b-galactosidase activity in cell lysate was assayed to evaluate the transcriptional function of p53 using Luminescent b-gal kit (BD Biosciences). Cell death assay and caspase activity assay. TPCK-treated HCT116 p53+/+ and HCT116 p53)/) cells were stained with trypan blue and the number of stained cells were counted using hemocytometer. HCT116 p53)/) cells grown on 35 mm culture dish were transfected with the wild-type p53 or mutant p53 (p53 5D) along with the plasmid encoding b-galactosidase. After incubation for 24 h, 50 lM TPCK was added and transfected cells were fixed and stained with 5-bromo-4chloro-3-indolyl-b-D -galactoside (X-gal). The induction of apoptosis in b-galactosidase positive blue cells expressing p53 derivatives was examined by microscopic observation. The results were expressed as mean values SE from the three independent experiments. HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and cell lysates containing the equal amount of total proteins (15 lg) were mixed with the assay buffer containing 93.7 mM Hepes (pH 7.5), 9.36% sucrose, 0.09% CHAPS, 10 mM DTT, 10 lg/ml leupeptin, and 5 lg/ml pepstatin. Cell lysate proteins were incubated with 200 lM of colorimetric peptide substrate, N-acetyl–Asp–Glu–Val–Asp p-nitroanilide (Ac-DEVD-pNA) for 2 h. The assay was performed at 37 °C and caspase activities were monitored by measuring the absorbance at 405 nm. Dephosphorylation of cellular p53. HCT116 p53+/+ cells (2 106 cells) were washed with PBS and incubated in a phosphate-free DMEM. After incubation for 1 h, cellular proteins were metabolically labeled by adding [32 P]orthophosphate (Amersham Biosciences) to the media (0.2 mCi/ml) and incubated for 4 h. TPCK was then treated for 30 min or 1 h before the lysis. Cell lysate proteins were immunoprecipitated using anti-p53 antibody (DO-1) and phosphorylation of p53 was examined by 10% SDS–PAGE and subsequent autoradiography.
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Results TPCK activates transcriptional and pro-apoptotic function of p53 The data in Fig. 1 demonstrate that TPCK induces p53-dependent cell death. HCT116 p53+/+ cells derived from human colorectal carcinoma underwent cell death after TPCK treatment. However, only the basal level of cell death was observed in TPCK treated HCT116 p53)/) cells under the same condition. The effects of TPCK on the transcriptional activity of p53 were evaluated by the activity of p21 promoter expressing b-galactosidase reporter (Fig. 1B). HCT116 p53)/) cells were transfected with p53 and the activity of p21 promoter was measured after TPCK treatment. The transcriptional activity of p21 promoter sequence linked to b-galactosidase reporter was almost threefold higher in cells incubated for 6 h after 50 lM TPCK treatment than in control cells which were not treated with TPCK (Fig. 1B). Stimulation of p21 expression by p53 trans-
Fig. 1. p53-Dependent induction of cell death by TPCK in HCT116 cells. (A) HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and cell viabilities were measured by trypan blue exclusion method. (B) HCT116 p53)/) cells were co-transfected with pGL2-b-gal containing p21 promoter, a control reporter plasmid (pGL-3-luc), and the expression plasmid encoding the full-length p53 (pcDNA-HA-p53). After incubation for 24 h, 25 or 50 lM TPCK was treated and incubated for additional hours. Cells were harvested and bgalactosidase activities in transfected cells were measured by Luminescent b-gal kit (BD Biosciences). (C) HCT116 p53+/+ cells were treated with 50 lM TPCK for the indicated amount of time. Cell lysates were analyzed by SDS–PAGE followed by immunoblotting using antibodies against actin, p53, and p21.
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activation was also examined in HCT116 p53+/+ cells after TPCK treatment. Cellular levels of p21 protein were significantly elevated in 3 h after TPCK treatment and maintained thereafter (Fig. 1C). Dephosphorylation of p53 by TPCK The effects of TPCK on the state of phosphorylation of p53 were examined in HCT116 p53+/+ cells (Fig. 2). The cellular levels of p53 in HCT116 p53+/+ cells were not affected by TPCK treatment. However, TPCK-induced dephosphorylation of p53 in HCT116 p53+/+ cells in which cellular proteins including p53 were metabolically labeled with 32 P. Immunoblot analysis using phospho-specific antibodies including P-Ser6, P-Ser9, P-Ser46, and p53 C-terminal specific antibody PAb421, which reacts with non-phosphorylated Ser376 and Ser378, indicates that the phosphorylation at serines 6, 9, 46, 376, and 378 was inhibited in HCT116 p53+/+ cells in response to TPCK treatment. Immunoblot analysis using the corresponding phospho-specific antibodies also revealed that the phosphorylation of p53 at serines 15, 20, 37, and 392 was unaffected by TPCK treatment (data not shown).
Fig. 2. TPCK-induced dephosphorylation of p53. (A) Dephosphorylation of p53 at serine residues by TPCK. TA, transactivation domain; DBD, DNA binding domain; and OD, oligomerization domain. (B) Cellular proteins in HCT116 p53+/+ cells (100 mm) were metabolically labeled with 32 P as described in ÔMaterials and methods.Õ Cellular p53 was immunoprecipitated and the incorporation of 32 P into cellular p53 was examined by 10% SDS–PAGE and autoradiography. (C) HCT116 p53+/+ cells were treated with 50 lM TPCK for the indicated amount of time and phosphorylation of serine residues on p53 in cell lysates proteins were examined by SDS–PAGE followed by immunoblotting using p53 specific phospho-amino acid antibodies. Note that PAb421 reacts with nonphosphorylated serine residues at 376 and 378.
p53-dependent caspase activation by TPCK Since caspases are executor molecules of apoptosis, it was reasonable to hypothesize that p53 might function to activate caspases in order to proceed p53-dependent apoptosis. Data in Fig. 3A implicate an indispensable role of p53 in the induction of apoptosis mediated by TPCK. TPCK treatment evoked caspase activation in HCT116 p53+/+ cells, but not in p53 deficient HCT116 p53)/) cells. Among the six caspases tested, at least caspase-3 and caspase-7 appeared to be activated in HCT116 p53+/+ cells in response to TPCK treatment. The activation of caspase-3 was also evidenced by the cleavage of cellular PARP in TPCK treated HCT116 p53+/+ cells. Both caspase-3 and caspase-7 were not activated by TPCK treatment in HCT116 p53)/) cells. Requirement of p53 dephosphorylation during cell death mediated by TPCK Knowing that TPCK induces dephosphorylation of serine residues on p53, it was necessary to determine whether the activation of transcriptional function of p53 and stimulation of apoptosis in TPCK treated HCT116
Fig. 3. Indispensable role of p53 in caspase activation. (A) HCT116 p53+/+ and HCT116 p53)/) cells were treated with 50 lM TPCK and caspase activities were measured using colorimetric peptide substrate. (B) The proteolytic cleavages of caspase-3, caspase-7, and PARP were analyzed by immunoblot analysis using antibodies against caspase-3, caspase-7, and PARP, respectively.
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p53+/+ cells were attributable to the dephosphorylation of serine residues on p53. The mutant p53, p53 5D, in which the potential dephosphorylation at serines 6, 9, 46, 376, and 378 was constitutively blocked by substituting serine residues by hydrophilic aspartic acids, was transfected into HCT116 p53)/) cells. Neither the activation of promoter activity of p21 nor the induction of apoptosis was observed in transfected cells expressing p53 5D after TPCK treatment (Fig. 4A). While caspase3 and caspase-7 were immediately activated and cleaved PARP in cells expressing wild-type p53 in response to 50 lM TPCK treatment, cells expressing p53 5D failed to trigger early apoptotic responses including caspase activation and PARP cleavage after TPCK treatment (Fig. 4B). Relatively high amount of cell death observed in p53 5D transfected cells at 9 h postincubation might reflect the cytotoxic potential of TPCK, which is an alkylating agent.
Discussion
Fig. 4. Effects of serine dephosphorylation in p53 on its transcription, TPCK-induced apoptosis, and caspase activation. (A) HCT116 p53)/) cells were transfected with wild-type p53 or mutant p53 5D and the activity of p21 promoter linked to the b-galactosidase reporter was measured after TPCK treatment. For apoptosis assay, HCT116 p53)/) cells were co-transfected with plasmid encoding b-galactosidase with p53 or p53 5D and cell death of transfected cells was measured after staining cells with 5-bromo-4-chloro-3-indolyl-b-D galactoside (X-gal). (B) HCT116 p53)/) cells were transfected with the empty vector (pcDNA3), wild-type p53 or mutant p53 5D, and treated with 50 lM TPCK for the indicated amount of time. Cell lysates were analyzed by immunoblot analysis using antibodies against caspase-3, caspase-7, and PARP.
Acknowledgments
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In this study, we initially report that TPCK (i) dephosphorylates the serine residues both at the C-terminal and N-terminal region of p53, (ii) stimulates the transcriptional activity of p53, and (iii) activates caspases to promote p53-dependent apoptosis in cultured human cells. Our data do not support the general claim that the phosphorylation of p53 increases its transcriptional function. The phosphorylation of p53 has been believed to be linked to the activation of its transcriptional function, protein stability, binding ability with other proteins or DNA, and state of oligomerization [11,13,16,19,21,22]. The biochemical links between the dephosphorylation at serine residues of p53 and the stimulation of apoptosis by TPCK have not been reported. However, there exist some implications that the dephosphorylation might be involved in the regulation of p53 function. Consistent with this speculation, the dephosphorylation of p53 at certain sites in the C-terminal domain after DNA damage has been demonstrated to enhance an association of p53 with 14-3-3 protein which activates p53 function [23,24]. Dephosphorylation of a serine residue in the N-terminal domain of p53 has also been reported in human melanoma A375 cell in response to DNA damage [25]. Our data presented in this study suggest that TPCK-induced apoptosis involves p53 dephosphorylation at serine residues. There are unsolved questions to be determined regarding this hypothesis. The roles of the dephosphorylation of serine residues in p53 in the signal transduction leading to the pro-apoptotic function demand further investigation. The involvement of dephosphorylation in TPCK-mediated apoptosis does not seem to be a restricted phenomenon that occurs in HCT116 cells derived from human colorectal carcinoma, since the dephosphorylation of p53 on serine residues was also observed in TPCK treated Cos cells derived from monkey kidney (data not shown). Our findings might provide some additional insights into the mechanisms whereby the state of p53 phosphorylation is modulated by TPCK to regulate signaling pathways influencing cell death and survival.
We thank Dr. John Blenis and Dr. Bryan Ballif for important communications and critical reading of the manuscript. This work was supported by the Grant 2002-CP0432 from Korea Research Foundation, South Korea.
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