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• W2052592595 abstract "K+ channel-associated protein/protein inhibitor of activated STAT (KChAP/PIAS3β) is a potassium (K+) channel modulatory protein that boosts protein expression of a subset of K+ channels and increases currents without affecting gating. Since increased K+efflux is an early event in apoptosis, we speculated that KChAP might induce apoptosis through its up-regulation of K+ channel expression. KChAP belongs to the protein inhibitor of activated STAT family, members of which also interact with a variety of transcription factors including the proapoptotic protein, p53. Here we report that KChAP induces apoptosis in the prostate cancer cell line, LNCaP, which expresses both K+ currents and wild-type p53. Infection with a recombinant adenovirus encoding KChAP (Ad/KChAP) increases K+ efflux and reduces cell size as expected for an apoptotic volume decrease. The apoptosis inducer, staurosporine, increases endogenous KChAP levels, and LNCaP cells, 2 days after Ad/KChAP infection, show increased sensitivity to staurosporine. KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15. Consistent with activation of p53 as a transcription factor, p21 levels are increased in infected cells. Wild-type p53 is not essential for induction of apoptosis by KChAP, however, since KChAP also induces apoptosis in DU145 cells, a prostate cancer cell line with mutant p53. Consistent with its proapoptotic properties, KChAP prevents growth of DU145 and LNCaP tumor xenografts in nude mice, indicating that infection with Ad/KChAP might represent a novel method of cancer treatment. K+ channel-associated protein/protein inhibitor of activated STAT (KChAP/PIAS3β) is a potassium (K+) channel modulatory protein that boosts protein expression of a subset of K+ channels and increases currents without affecting gating. Since increased K+efflux is an early event in apoptosis, we speculated that KChAP might induce apoptosis through its up-regulation of K+ channel expression. KChAP belongs to the protein inhibitor of activated STAT family, members of which also interact with a variety of transcription factors including the proapoptotic protein, p53. Here we report that KChAP induces apoptosis in the prostate cancer cell line, LNCaP, which expresses both K+ currents and wild-type p53. Infection with a recombinant adenovirus encoding KChAP (Ad/KChAP) increases K+ efflux and reduces cell size as expected for an apoptotic volume decrease. The apoptosis inducer, staurosporine, increases endogenous KChAP levels, and LNCaP cells, 2 days after Ad/KChAP infection, show increased sensitivity to staurosporine. KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15. Consistent with activation of p53 as a transcription factor, p21 levels are increased in infected cells. Wild-type p53 is not essential for induction of apoptosis by KChAP, however, since KChAP also induces apoptosis in DU145 cells, a prostate cancer cell line with mutant p53. Consistent with its proapoptotic properties, KChAP prevents growth of DU145 and LNCaP tumor xenografts in nude mice, indicating that infection with Ad/KChAP might represent a novel method of cancer treatment. Apoptosis, or programmed cell death, is a multistage process starting with cell shrinkage followed by chromatin condensation, caspase activation, and cellular fragmentation with subsequent removal of apoptotic bodies by neighboring cells. Early cell shrinkage is due in part to increased K+ efflux (reviewed in Ref. 1Yu S.P. Canzoniero L. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (329) Google Scholar). Loss of K+ during apoptosis is not an epiphenomenon but is critical to its progression. Block of K+ currents by channel-specific drugs or high extracellular K+ prevents apoptosis (2Yu S.P. Yeh C.H. Sensi S.L. Gwag B.J. Canzoniero L.M. Farhangrazi Z.S. Ying H.S. Tian M. Dugan L.L. Choi D.W. Science. 1997; 278: 114-117Crossref PubMed Scopus (532) Google Scholar, 3Yu S.P. Yeh C.H. Gottron F. Wang X Grabb M.C. Choi D.W. J. Neurochem. 1999; 73: 933-941Crossref PubMed Scopus (129) Google Scholar, 4Krick S. Platoshyn O. Sweeney M. Kim H. Yuan J.X. Am. J. Physiol. 2001; 280: C970-C979Crossref PubMed Google Scholar, 5Krick S. Platoshyn O. McDaniel S.S. Rubin L.J. Yuan J.X. Am. J. Physiol. 2001; 281: L887-L894Crossref PubMed Google Scholar). Importantly, caspase activation, considered the point of no return in apoptosis, only occurs after cellular K+loss (6Hughes F.M. Cidlowski J. Adv. Enzyme Reg. 1999; 39: 157-171Crossref PubMed Scopus (202) Google Scholar). Despite the importance of this process, virtually nothing is known about the mechanisms generating increased K+ efflux. Here, we investigate the link between the actions of a K+channel modulatory protein, KChAP/PIAS3β, 1The abbreviations used are: KChAPK+ channel-associated proteinPIASprotein inhibitor of activated STATSTATsignal transducers and activators of transcriptionSTSstaurosporineAdadenovirusGFPgreen fluorescent proteinPBSphosphate-buffered salineMOImultiplicity of infectionPIpropidium iodidepotassium-binding benzofuran isophthalateTUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingPARPpoly(ADP-ribose) polymerase 1The abbreviations used are: KChAPK+ channel-associated proteinPIASprotein inhibitor of activated STATSTATsignal transducers and activators of transcriptionSTSstaurosporineAdadenovirusGFPgreen fluorescent proteinPBSphosphate-buffered salineMOImultiplicity of infectionPIpropidium iodidepotassium-binding benzofuran isophthalateTUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelingPARPpoly(ADP-ribose) polymerase and apoptosis in tumor cells.KChAP/PIAS3β is a K+ channel modulatory protein that exhibits “chaperone-like” behavior toward a subset of K+ channels (7Wible B.A. Yang Q. Kuryshev Y.A. Accili E.A. Brown A.M. J. Biol. Chem. 1998; 273: 11745-11751Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Kuryshev Y.A. Gudz T.I. Brown A.M. Wible B.A. Am. J. Physiol. 2000; 278: C931-C941Crossref PubMed Google Scholar, 9Kuryshev Y.A. Wible B.A. Gudz T.I. Ramirez A.N. Brown A.M. Am. J. Physiol. 2001; 281: C290-C299Crossref Google Scholar). KChAP, a soluble protein, binds transiently to the cytoplasmic NH2 termini of its target channels and increases channel expression in a transcription-independent manner. Both total channel protein and surface expression are increased in response to KChAP. We hypothesized that the increased K+ channel expression conferred by KChAP might be associated with apoptosis.In addition to K+ channels, KChAP/PIAS3β interacts with other binding partners, most notably a variety of transcription factors. KChAP belongs to the protein inhibitor of activated STAT (PIAS) gene family. There are four mammalian members of this family: 1) KChAP (7Wible B.A. Yang Q. Kuryshev Y.A. Accili E.A. Brown A.M. J. Biol. Chem. 1998; 273: 11745-11751Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar)/PIAS3 (10Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (796) Google Scholar), 2) Gu-binding protein (11Valdez B.C. Henning D. Perlaky L. Busch R.K. Busch H. Biochem. Biophys. Res. Commun. 1997; 234: 335-340Crossref PubMed Scopus (54) Google Scholar)/PIAS1 (12Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (627) Google Scholar), 3) androgen receptor-interacting protein 3 (13Moilanen A.M. Karvonen U. Poukka H. Yan W. Toppari J. Janne O.A. Palvimo J.J. J. Biol. Chem. 1999; 274: 3700-3704Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar)/PIASxα (12Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (627) Google Scholar) and Miz1 (14Wu L., Wu, H., Ma, L. Sangiorgi F., Wu, N. Bell J.R. Lyons G.E. Maxson R. Mech. Dev. 1997; 65: 3-17Crossref PubMed Scopus (89) Google Scholar)/PIASxβ (12Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (627) Google Scholar), and 4) PIASy (12Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (627) Google Scholar, 15Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar). The multiple names of most of these genes reflect their independent cloning as binding partners of different proteins. KChAP and PIAS3 are alternatively spliced products of a single gene; in KChAP, a small intron in the NH2-terminal coding region is retained, generating an in-frame insertion of 39 amino acids. We refer to KChAP as PIAS3β to distinguish it from the original mouse PIAS3 clone (10Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (796) Google Scholar), which we refer to as PIAS3α. Mouse PIAS3 (PIAS3α) binds to activated STAT3 and prevents its attachment to DNA (10Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (796) Google Scholar). PIAS proteins may also act as coregulators of steroid hormone transcription factors including androgen, glucocorticoid, and progesterone receptors (13Moilanen A.M. Karvonen U. Poukka H. Yan W. Toppari J. Janne O.A. Palvimo J.J. J. Biol. Chem. 1999; 274: 3700-3704Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16Kotaja N. Aittomaki S. Silvennoinen O. Palvimo J.J. Janne O.A. Mol. Endocrinol. 2000; 14: 1986-2000Crossref PubMed Scopus (145) Google Scholar, 17Tan J. Hall S.H. Hamil K.G. Grossman G. Petrusz P. Liao J. Shuai K. French F.S. Mol. Endocrinol. 2000; 14: 14-26Crossref PubMed Scopus (86) Google Scholar, 18Gross M. Liu B. Tan J. French F.S. Carey M. Shuai K. Oncogene. 2001; 20: 3880-3887Crossref PubMed Scopus (147) Google Scholar). PIASy (15Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar) and PIAS1 (19Megidish T. Xu J.H. Xu C.W. J. Biol. Chem. 2002; 277: 8255-8259Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) have been found to interact with p53 and differentially affect its transcriptional activity. PIASy blocked the ability of p53 to transcribe its target gene, p21 (15Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar), whereas PIAS1 activated p53-mediated gene expression including p21 (19Megidish T. Xu J.H. Xu C.W. J. Biol. Chem. 2002; 277: 8255-8259Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).The interactions of PIAS proteins (KChAP/PIAS3β) with K+channels and p53 were the starting point of the present experiments. We hypothesized that KChAP might contribute to apoptosis, on the one hand by increasing K+ efflux in association with apoptotic cell volume decrease and, on the other, by activating p53. To test this hypothesis, a nonreplicating, recombinant adenovirus containing KChAP cDNA was constructed (AdKChAP) for infection of LNCaP cells, selected because they express both K+ currents and wild type p53. We found that overexpression of KChAP increased K+ efflux, reduced LNCaP cell volume, and induced apoptosis as evidenced by positive COMET assay and PARP cleavage. KChAP interacted with the p53 tetramerization domain in yeast two-hybrid studies, and total p53 as well as the phosphoserine 15 form were increased in Ad/KChAP-infected LNCaP cells. Consistent with activation of p53 transcription factor activity, p21, a G1 cell cycle arrest protein, was up-regulated in these cells. Ad/KChAP also produced apoptosis in another prostate cancer cell line, DU145 cells, which contain mutant p53. Given its proapoptotic effects independent of p53 status, we proposed that KChAP might act as a tumor suppressor, and we found that injection of Ad/KChAP into LNCaP and DU145 tumor xenografts in nude mice produced apoptosis and suppression of tumor growth.EXPERIMENTAL PROCEDURESCell Culture and Adenovirus InfectionLNCaP, DU145, and Jurkat cells were obtained from the American Type Culture collection. LNCaP and Jurkat cells were maintained in RPMI medium with 10% fetal bovine serum, while DU145 cells were propagated in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. All media also contained 100 units/ml penicillin and 100 μg/ml streptomycin. In the LNCaP experiments with high extracellular K+ in the medium, RPMI medium was assembled from the individual components as outlined by Invitrogen so that we could adjust the [K+]. The total amount of K+ plus Na+ in the media was kept constant at 108.4 mm so that when [K+] was elevated, [Na+] was correspondingly decreased. Staurosporine (STS) was from Sigma, and a 1 mm stock solution was prepared in Me2SO and stored at −20 °C. A final concentration of 1 μm was used to induce apoptosis.A replication-defective, recombinant KChAP/adenovirus was constructed as follows. Full-length KChAP cDNA was subcloned in the vector, pShuttle-CMV, and sent to Q-Biogene (Montreal, Quebec, Canada) for adenovirus construction and purification. Expression of KChAP from the recombinant adenovirus, Ad/KChAP, was verified by Western blotting lysates of infected cells with a KChAP-specific antibody, 088, which recognizes only overexpressed KChAP (see details below). Recombinant Ad/GFP and Ad/LacZ were purchased from Q-Biogene. Viral infections were performed by diluting the virus to the appropriate concentration in standard medium and overlaying the cells (1 ml/35-mm dish). The medium was not changed before the cells were harvested.Antibodies and Western BlottingWe used two KChAP antibodies in this study, both of which were generated in our laboratory. 899 was raised against a bacterial fusion protein that consisted of the COOH-terminal 169 amino acids of KChAP (5Krick S. Platoshyn O. McDaniel S.S. Rubin L.J. Yuan J.X. Am. J. Physiol. 2001; 281: L887-L894Crossref PubMed Google Scholar). It recognizes both endogenous and overexpressed KChAP. 088 was raised against a peptide in the NH2 terminus of KChAP that is not present in PIAS3 (SPSPLASIPPTLLTPGTLLGPKREVDMH), hence the PIAS3α and PIAS3β/KChAP nomenclature used here. 088 recognizes overexpressed but not endogenous KChAP. Affinity-purified antibodies were used in Western blotting. Other antibodies used for Western blotting to detect the following proteins were obtained from commercial sources: p53 (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); STAT1, STAT3, and cyclins A, B, and D3 (Transduction Laboratories, Lexington, KY); actin (clone AC-40; Sigma); phospho-p53 (Ser15) (Cell Signaling Technology, Inc., Beverly, MA); PARP (we used two antibodies interchangeably that recognize both intact and cleaved PARP, one from Cell Signaling Inc. and one from BD PharMingen (San Diego, CA)); monoclonal Rb (BD PharMingen); and p21 (WAF1 Ab1; Oncogene Research Products (Boston, MA)).Cells were lysed in a buffer consisting of 1% Triton X-100, 150 mm NaCl, 50 mm Tris, 1 mm EDTA, pH 7.5 containing freshly added protease inhibitors (Complete; Roche Molecular Biochemicals) and the phosphatase inhibitors sodium fluoride (50 mm) and sodium orthovanadate (1 mm) for 30 min on ice. Insoluble debris was pelleted at 20,800 ×g for 10 min at 4 °C. Lysate protein concentrations were determined by the BCA method (Pierce), and aliquots were boiled in a reducing SDS sample buffer to denature protein. SDS-PAGE gels were blotted to polyvinylidene difluoride membranes using a semidry blotting apparatus. Blots were blocked overnight in 5% milk (Bio-Rad) in PBS-T (PBS plus 0.1% Tween 20) at 4 °C. Primary antibodies diluted in blocking buffer were incubated with the blots for 1 h at room temperature. Blots were washed with PBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (AmershamBiosciences) in blocking buffer for 1 h at room temperature. Blots were developed with the ECL-Plus kit (Amersham Biosciences).Yeast Two-hybrid AssayFull-length KChAP (residues 1–619) in the GAL4 activation domain vector, pGAD424, was used as described (5Krick S. Platoshyn O. McDaniel S.S. Rubin L.J. Yuan J.X. Am. J. Physiol. 2001; 281: L887-L894Crossref PubMed Google Scholar). Murine p53 (residues 90–390) in a GAL4 DNA binding domain vector was from the CLONTECH Matchmaker yeast two-hybrid kit. The murine p53 carboxyl terminus (residues 290–390) and subfragments (Phe324–Thr352) and (Phe334–Thr352) with EcoRI and SalI sites incorporated at the 5′- and 3′-ends of the fragments, respectively, were generated by PCR. PCR products were subcloned using TOPO cloning (Invitrogen; Carlsbad, CA) and sequenced before cloning in frame into pGBT9 and pGAD424. Yeast strain Y190 was transformed with combinations of pGBT9 and pGAD424 plasmids, and interaction was determined by β-galactosidase filter assays as previously described (7Wible B.A. Yang Q. Kuryshev Y.A. Accili E.A. Brown A.M. J. Biol. Chem. 1998; 273: 11745-11751Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).COMET AssayDNA degradation was assayed in cells overexpressing Ad/KChAP or Ad/LacZ using the kit from Trevigen. Briefly, 106 cells/ml were mixed with molten low melting agarose at a ratio of 1:10. Immediately, 50 μl of this mixture was spread onto slides. Slides were immersed in prechilled lysis solution at 4 °C for 30 min. After a brief rinse in 1× TBE, the slides were subjected to horizontal electrophoresis at 1 V/cm (measured electrode to electrode) for 11 min. The slides were then put in ice-cold methanol for 5 min followed by a 5-min incubation at room temperature in ethanol. After drying, the slides were stained with SYBR green for epifluorescence microscopy.Rb+ FluxLNCaP cells were plated in six-well tissue culture dishes at 250,000 cells/well. On the following day, cells were infected with either Ad/GFP or Ad/KChAP (multiplicity of infection (MOI) = 100). Rb+ fluxes were measured 24 h after infection using the nonradioactive method of Terstappen (20Terstappen G.C. Anal. Biochem. 1999; 272: 149-155Crossref PubMed Scopus (93) Google Scholar). To load Rb+, cells were incubated for 4 h (37 °C) in a modified Tyrode's solution containing 5 mm RbCl, 145 mm NaCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm glucose (pH 7.4 at 37 °C), and 10% fetal bovine serum. The cells were then washed three times with Rb+-free PBS and incubated for 10 min at room temperature in 1 ml of normal Tyrode's solution. The supernatant containing released Rb+ was collected, and the cells were lysed in 1 ml of PBS containing 1% Triton X-100 to measure Rb+ remaining in the cells. Samples were diluted (1:4) with ionization buffer (PBS containing 2.5% HNO3), and Rb+ content was determined using flame atomic absorption spectrometry at 780 nm (PerkinElmer Life Sciences model 3100). A calibration curve was constructed to determine Rb+concentrations. Relative Rb+ efflux was calculated as the amount of Rb+ in the supernatant divided by total Rb+ (supernatant plus cell lysate).Flow Cytometric AnalysisK+ ContentAt 72 h postinfection with either Ad/GFP or Ad/KChAP (MOI = 100), LNCaP cells were collected by trypsin treatment and washed in PBS. The K+-sensitive dye, potassium-binding benzofuran isophthalate (PBFI) (Molecular Probes, Inc., Eugene, OR) was dissolved in Pluronic F-127 (Molecular Probes) and incubated with the cells in standard medium at a final concentration of 5 μm for 1 h at 37 °C. The cells were then chilled on ice, and propidium iodide (5 μg/ml) was added. Flow cytometry was performed with a Becton Dickinson FACS Vantage machine. Ten thousand cells from each treatment group were analyzed. Excitation of PBFI was at 340 nm, and emission was captured at 425 nm. Propidium iodide was excited by a 488-nm argon laser at the same time.DNA ContentFor analysis of DNA content, cells were trypsinized either 24 or 72 h postinfection as described above, washed with PBS, and fixed in cold 70% ethanol for at least 8 h at −20 °C. After washing in PBS, propidium iodide (5 μg/ml) was added. Ten thousand cells were examined by flow cytometry for each sample using a Becton Dickinson FACScan (excitation at 488 nm).Tumor Production and Adenovirus Injection in Nude MiceTumor cells (DU145 or LNCaP; 2 × 106cells/injection site) were suspended in serum-free Dulbecco's modified Eagle's medium, mixed with an equal volume of cold Matrigel on ice, and injected subcutaneously into both flanks of 8–9-week-old female BALB/c nude mice. Tumor growth was monitored using calipers every 2–3 days. Tumor volume was calculated as (L ×W 2)/2, where L represents length and W is width in millimeters. When tumors reached an average size of 50–60 mm3 (about 2 weeks for DU145 and 5 weeks for LNCaP), mice were divided into three treatment groups: 1) PBS, 2) Ad/GFP, and 3) Ad/KChAP. Both tumors on an individual mouse received the same treatment. Ad/GFP and Ad/KChAP were diluted in sterile PBS to 5 × 108 plaque-forming units/μl. Injections (1 μl/mm3 of tumor) were delivered directly into the tumors every 2–3 days for a total of three injections per week. Assuming 106 cells per mm3 of tumor, about 500 plaque-forming units of virus per tumor cell was injected at 48–72 h intervals. Mice were sacrificed by cervical dislocation 48 h after the final injection, and tumors were dissected and frozen in liquid nitrogen. During the experiments, the animals were housed and handled in accordance with the National Institutes of Health guidelines.Immunohistochemistry and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay of Tumor SectionsEight-micron sections were prepared from frozen tumors dissected from the three treatment groups (PBS, Ad/GFP, and Ad/KChAP), mounted, and fixed on glass slides. Overexpressed KChAP was detected by incubating sections with the 088 antibody (1:100 dilution in 0.2% gelatin plus 0.5% bovine serum albumin/PBS) for 2 h at room temperature, washing with PBS, and incubating with biotinylated anti-rabbit secondary antibody (1:200) for 1 h at room temperature. Color development was done with the ABC and DAB kits from Vector Laboratories following their instructions. Apoptosis of cells in tumors subjected to different treatments was determined by the TUNEL assays using the Apo-Tag kit (Oncor, Inc.), following the manufacturer's instructions.DISCUSSIONWe have shown that infection of a recombinant adenovirus overexpressing KChAP/PIAS3β (Ad/KChAP) in LNCaP and DU145 prostate cancer cells produces apoptosis, and direct injection of Ad/KChAP into xenografts of LNCaP and DU145 cells in nude mice suppresses tumor growth. These data support a link between overexpression of KChAP/PIAS3β and the increased K+ efflux that results during apoptotic volume decrease. Increased K+ efflux from Ad/KChAP-infected LNCaP cells was shown by Rb+ flux measurements, and decreased intracellular K+ and cell shrinkage were demonstrated by flow cytometry. Apoptosis in Ad/KChAP-infected LNCaP cells was blocked by high extracellular K+, indicating that KChAP-enhanced K+ efflux is critical for apoptosis. The identity of the K+ channel(s) that carries the increased outward current has not yet been determined. We attempted to infect cells in the presence of K+ channel blockers such as 4-AP, TEA, and quinidine to assess the effects on apoptosis, but these experiments were unsuccessful, since the drugs also interfered with viral infection. LNCaP cells are known, however, to possess several outward K+ currents including voltage-gated delayed rectifier channels (30Skyrma R.N. Prevarskaya N.B. Dufy-Barbe L. Odess M.F. Audin J. Dufy B. Prostate. 1997; 33: 112-122Crossref PubMed Scopus (95) Google Scholar, 31Laniado M.E. Fraser S.P. Djamgoz M.B. Prostate. 2001; 46: 262-274Crossref PubMed Scopus (64) Google Scholar) as well as several two-pore K+ channel-like currents.2 Recently, two-pore K+ channels were proposed as candidates for mediating apoptotic volume decrease (32Trimarchi J.R. Liu L. Smith P.J.S. Keefe D.L. Am. J. Physiol. 2002; 282: C588-C594Crossref PubMed Scopus (77) Google Scholar). Future experiments should reveal both the K+ channels mediating apoptotic volume decrease in LNCaP cells and the role of KChAP in modulating these channels. With the results presented here, we propose that KChAP is an attractive candidate for the long sought after mediator of apoptotic volume decrease.KChAP/PIAS3β not only affects K+ channels but, as a member of the PIAS family, interacts with a variety of transcription factors. We have found that KChAP/PIAS3β, like other members of the PIAS family, binds to p53 and alters its transcriptional activity. Interestingly, the same KChAP domain that binds to Kv channel NH2 termini (i.e. the 98-amino acid M-fragment) (8Kuryshev Y.A. Gudz T.I. Brown A.M. Wible B.A. Am. J. Physiol. 2000; 278: C931-C941Crossref PubMed Google Scholar) also interacts with p53 in yeast two-hybrid experiments. We have identified a 28-residue fragment from the COOH terminus of p53, consisting of the tetramerization domain, which is sufficient for interaction with KChAP. This result is consistent with a recent report indicating that PIAS1 also interacts with the tetramerization domain of p53 (19Megidish T. Xu J.H. Xu C.W. J. Biol. Chem. 2002; 277: 8255-8259Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).Our data show that KChAP/PIAS3β activates p53 in LNCaP cells. When p53 is activated as a transcription factor, the cell cycle arrest protein gene, p21, is a target. We see both increased total p53 and p21 levels in Ad/KChAP-infected LNCaP cells, suggesting an activation of p53. PIAS1 has been shown to interact with p53 and activate its transcriptional activity (19Megidish T. Xu J.H. Xu C.W. J. Biol. Chem. 2002; 277: 8255-8259Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Another PIAS family member, PIASy, interacts with p53 but depresses rather than enhances its activity as a transcription factor (15Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar), suggesting distinct functions of individual PIAS proteins.One mechanism whereby PIAS proteins might activate p53 is suggested by our observation of increased p53 phosphorylation on serine 15 in Ad/KChAP-infected cells. Phosphorylation of this residue has been shown previously to correlate with increased stability of p53 as well as increased transcriptional activity (24Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar, 25Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar). KChAP-induced loss of K+ is not required for p53 activation, since serine 15 phosphorylation is also detected in cells bathed in high extracellular K+. This effect is distinct from caspase activation, which requires loss of intracellular K+. It is not known whether other PIAS proteins induce the same modification in p53 or how KChAP produces this post-translational modification. Several kinases have been implicated in the phosphorylation of p53 serine 15 including ATM, ATR, and c-Jun N-terminal kinase (reviewed in Ref. 33Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar). Interestingly, PIAS1 has been shown to induce apoptosis in U2OS cells via activation of c-Jun N-terminal kinase (34Liu B. Shuai K. J. Biol. Chem. 2001; 276: 36624-36631Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), suggesting a possible link between c-Jun N-terminal kinase and p53 phosphorylation. We have observed activation of c-Jun N-terminal kinase as well as p38 and extracellular signal-regulated kinase 1 and 2 kinases in Ad/KChAP-infected LNCaP cells.2The pleiotropic nature of PIAS protein action as well as the variety of binding partners identified thus far must now be considered in light of the recent implication of PIAS proteins as SUMO-1 E3 ligases. PIAS1 has been shown to catalyze the sumoylation (i.e. covalent attachment of the small ubiquitin-like modifier protein, SUMO-1) of p53 in mammalian cells (35Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar), and interestingly, sumoylation was previously reported to increase the transcriptional activity of p53 (36Gostissa M. Hengstermannm A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (437) Google Scholar, 37Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (558) Google Scholar). In Saccharomyces cerevesiae, the PIAS homolog, Siz1, has been shown to act as an E3-like factor for SUMO-1 conjugation to the septins, a process required for yeast budding (38Johnson E.S. Gupta A.A. Cell. 2001; 106: 735-744Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 39Takahashi Y. Toh-e A. Kikuchi Y. Gene (Amst.). 2001; 275: 223-231Crossref PubMed Scopus (107) Google Scholar, 40Takahashi Y. Kahyo T. Toh-e A. Yasuda H. Kikuchi Y. J. Biol. Chem. 2001; 276: 48973-48977Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In addition, the transcriptional activity of the androgen receptor, another PIAS-binding protein, can be modified by sumoylation (41Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (368) Google Scholar), although it is not known whether PIAS proteins mediate this process. It will be important to determine the role that sumoylation plays in the involvement of PIAS proteins with all binding partners, including the interaction of KC" @default.
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• W2052592595 title "Increased K+ Efflux and Apoptosis Induced by the Potassium Channel Modulatory Protein KChAP/PIAS3β in Prostate Cancer Cells" @default.
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