PHAPI/pp32 Suppresses Tumorigenesis by Stimulating Apoptosis
2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês
10.1074/jbc.m805801200
ISSN1083-351X
AutoresWei Pan, Li S. da Graca, Yufang Shao, Qian Yin, Hao Wu, Xuejun Jiang,
Tópico(s)Ubiquitin and proteasome pathways
ResumoPHAPI/pp32 is a tumor suppressor whose expression is altered in various human cancers. Although PHAPI possesses multiple biochemical activities, the molecular basis for its tumor-suppressive function has remained obscure. Recently we identified PHAPI as an apoptotic enhancer that stimulates apoptosome-mediated caspase activation. In this study, we defined the structural requirement for its activity to stimulate caspase activation using a series of truncation mutants of PHAPI. Further, utilizing these mutants, we provide evidence to support the model that the apoptotic activity of PHAPI is required for its tumor-suppressive capability. Consistently, pp32R1, a close homolog of PHAPI and yet an oncoprotein, is not able to stimulate caspase activation. A highly discrete region between these two proteins localizes to an essential caspase activation motif of PHAPI. Additionally, PHAPI is predominantly a nuclear protein, and it can translocate to the cytoplasm during apoptosis. Disruption of the nuclear localization signal of PHAPI caused a modest decrease of its tumor-suppressive function, indicating that nuclear localization of PHAPI contributes to, but is not essential for, tumor suppression. PHAPI/pp32 is a tumor suppressor whose expression is altered in various human cancers. Although PHAPI possesses multiple biochemical activities, the molecular basis for its tumor-suppressive function has remained obscure. Recently we identified PHAPI as an apoptotic enhancer that stimulates apoptosome-mediated caspase activation. In this study, we defined the structural requirement for its activity to stimulate caspase activation using a series of truncation mutants of PHAPI. Further, utilizing these mutants, we provide evidence to support the model that the apoptotic activity of PHAPI is required for its tumor-suppressive capability. Consistently, pp32R1, a close homolog of PHAPI and yet an oncoprotein, is not able to stimulate caspase activation. A highly discrete region between these two proteins localizes to an essential caspase activation motif of PHAPI. Additionally, PHAPI is predominantly a nuclear protein, and it can translocate to the cytoplasm during apoptosis. Disruption of the nuclear localization signal of PHAPI caused a modest decrease of its tumor-suppressive function, indicating that nuclear localization of PHAPI contributes to, but is not essential for, tumor suppression. Apoptosis plays a crucial role in multiple physiological events, and its deregulation can lead to various human diseases, including cancer, neurodegeneration, and autoimmune disorders (1Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12874) Google Scholar, 2Horvitz H.R. Cancer Res. 1999; 59: 1701s-1706sPubMed Google Scholar). Apoptosis is executed by a family of proteases known as caspases (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar, 4Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2269) Google Scholar). In mammals, a major caspase activation pathway is the mitochondrial cytochrome c-mediated caspase activation pathway, also known as the intrinsic apoptotic pathway (5Jiang X. Wang X. Annu. Rev. Biochem. 2004; 73: 87-106Crossref PubMed Scopus (1128) Google Scholar). In this pathway, a variety of apoptotic stimuli induce release of cytochrome c from mitochondria to the cytoplasm, a process controlled by the Bcl-2 family members (6Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Crossref PubMed Scopus (1517) Google Scholar, 7Harris M.H. Thompson C.B. Cell Death Differ. 2000; 7: 1182-1191Crossref PubMed Scopus (438) Google Scholar, 8Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). The released cytochrome c binds to Apaf-1, the essential mediator of the pathway, and triggers its nucleotide binding/exchanging activity (9Jiang X. Wang X. J. Biol. Chem. 2000; 275: 31199-31203Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 10Kim H.E. Du F. Fang M. Wang X. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17545-17550Crossref PubMed Scopus (238) Google Scholar). Subsequently, the activated Apaf-1 protein forms a multimeric protein complex called the apoptosome, which in turn recruits and activates the initiator caspase, caspase-9. The active apoptosome-caspase-9 holoenzyme (9Jiang X. Wang X. J. Biol. Chem. 2000; 275: 31199-31203Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 11Rodriguez J. Lazebnik Y. Genes Dev. 1999; 13: 3179-3184Crossref PubMed Scopus (461) Google Scholar) then cleaves and activates the effector caspases, including caspase-3 and caspase-7, and therefore causes apoptotic cell death. The cytochrome c-mediated caspase activation pathway is under precise regulation. For example, in the upstream of mitochondria, there are both pro-apoptotic and anti-apoptotic Bcl-2 family proteins that dictate mitochondrial cytochrome c release (6Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Crossref PubMed Scopus (1517) Google Scholar, 7Harris M.H. Thompson C.B. Cell Death Differ. 2000; 7: 1182-1191Crossref PubMed Scopus (438) Google Scholar, 8Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). In the downstream of mitochondria, inhibitor of apoptosis proteins negatively regulate apoptosis by inhibiting caspase activity (12Duckett C.S. Nava V.E. Gedrich R.W. Clem R.J. Van Dongen J.L. Gilfillan M.C. Shiels H. Hardwick J.M. Thompson C.B. EMBO J. 1996; 15: 2685-2694Crossref PubMed Scopus (523) Google Scholar, 13Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. 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Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar), which are also released from mitochondria during apoptosis. Further, nucleotide binding/exchanging of Apaf-1, a key biochemical event of the pathway, is also closely regulated. Recently, we found that the Apaf-1-dependent caspase activation can be enhanced by PHAPI (putative HLA-associated protein-I, also known as pp32) (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). Biochemically, PHAPI along with cellular apoptosis susceptibility and the heat shock protein HSP70, function as the nucleotide exchange factor for Apaf-1, a mechanism reminiscent of G protein regulation (21Kim H.E. Jiang X. Du F. Wang X. Mol. Cell. 2008; 30: 239-247Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Interestingly, PHAPI is predominantly a nuclear protein (22Chen T.H. Brody J.R. Romantsev F.E. Yu J.G. Kayler A.E. Voneiff E. Kuhajda F.P. Pasternack G.R. Mol. Biol. Cell. 1996; 7: 2045-2056Crossref PubMed Scopus (78) Google Scholar) and can translocate to the cytoplasm during apoptosis, a process involving the RNA-binding protein HuR (23Mazroui R. Di Marco S. Clair E. von Roretz C. Tenenbaum S.A. Keene J.D. Saleh M. Gallouzi I.E. J. Cell Biol. 2008; 180: 113-127Crossref PubMed Scopus (98) Google Scholar). Therefore, subcellular translocation of PHAPI provides an additional regulatory mechanism for PHAPI-stimulated caspase activation. In addition to its activity to promote apoptosis, PHAPI has been shown to possess other biochemical activities, including inhibition of protein phosphatase 2A (PP2A) 3The abbreviations used are: PP2A, protein phosphatase 2A; HAT, histone acetyltransferase; pp32R1, pp32-related-1; NLDM, nuclear localization defective mutant of PHAPI; CMV, cytomegalovirus; GFP, green fluorescent protein; WT, wild type; PRP, PHAPI recombinant protein with residues 140–163 swapped with the correspondent residues of pp32R1. and histone acetyltransferase (HAT), both crucial regulators for various physiological processes (24Li M. Makkinje A. Damuni Z. Biochemistry. 1996; 35: 6998-7002Crossref PubMed Scopus (171) Google Scholar, 25Seo S.B. Macfarlan T. McNamara P. Hong R. Mukai Y. Heo S. Chakravarti D. J. Biol. Chem. 2002; 277: 14005-14010Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). PHAPI can also interact with Ataxin-1, a protein mutated in the neurodegenerative disease spinocerebellar ataxia type I (26Matilla A. Koshy B.T. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar), suggesting a possible role of PHAPI in this type of neurodegeneration. The multiple biochemical activities of PHAPI suggest that this protein is involved in diverse biological events. To date, the most defined biological function of PHAPI is its role as a tumor suppressor. PHAPI expression appears to be greatly decreased during pancreatic cancer progression (27Brody J.R. Witkiewicz A. Williams T.K. Kadkol S.S. Cozzitorto J. Durkan B. Pasternack G.R. Yeo C.J. Mod. Pathol. 2007; 20: 1238-1244Crossref PubMed Scopus (19) Google Scholar). In a study of human non-small cell lung cancer, PHAPI expression level correlated with improved outcome following chemotherapy (28Hoffarth S. Zitzer A. Wiewrodt R. Hahnel P.S. Beyer V. Kreft A. Biesterfeld S. Schuler M. Cell Death Differ. 2008; 15: 161-170Crossref PubMed Scopus (39) Google Scholar). Functionally, overexpression of PHAPI can inhibit oncogene-induced tumorigenesis (22Chen T.H. Brody J.R. Romantsev F.E. Yu J.G. Kayler A.E. Voneiff E. Kuhajda F.P. Pasternack G.R. Mol. Biol. Cell. 1996; 7: 2045-2056Crossref PubMed Scopus (78) Google Scholar, 29Brody J.R. Kadkol S.S. Mahmoud M.A. Rebel J.M. Pasternack G.R. J. Biol. Chem. 1999; 274: 20053-20055Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 30Bai J. Brody J.R. Kadkol S.S. Pasternack G.R. Oncogene. 2001; 20: 2153-2160Crossref PubMed Scopus (64) Google Scholar). Intriguingly, PHAPI has a close homolog known as pp32R1 (pp32-related-1), which is an oncoprotein instead of a tumor suppressor (29Brody J.R. Kadkol S.S. Mahmoud M.A. Rebel J.M. Pasternack G.R. J. Biol. Chem. 1999; 274: 20053-20055Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Kadkol S.S. Brody J.R. Pevsner J. Bai J. Pasternack G.R. Nat. Med. 1999; 5: 275-279Crossref PubMed Scopus (5) Google Scholar). The expression of pp32R1 is switched on in certain human cancers with diminished PHAPI expression (31Kadkol S.S. Brody J.R. Pevsner J. Bai J. Pasternack G.R. Nat. Med. 1999; 5: 275-279Crossref PubMed Scopus (5) Google Scholar, 32Kadkol S.S. El Naga G.A. Brody J.R. Bai J. Gusev Y. Dooley W.C. Pasternack G.R. Breast Cancer Res. Treat. 2001; 68: 65-73Crossref PubMed Scopus (26) Google Scholar). The biochemical activity of PHAPI responsible for its tumor-suppressive function has not been defined. Although both PP2A and HAT have been implicated in cancer development, whether PHAPI is able to suppress tumorigenesis via inhibiting these two enzymes is not clear. On the other hand, the apoptosis-accelerating activity of PHAPI provides a plausible explanation for its tumor-suppressive role, which is consistent with recent studies showing that in human breast cancer and non-small cell lung cancer, PHAPI expression level correlates with the sensitivity of cancer cells to Apaf-1-mediated apoptosis (28Hoffarth S. Zitzer A. Wiewrodt R. Hahnel P.S. Beyer V. Kreft A. Biesterfeld S. Schuler M. Cell Death Differ. 2008; 15: 161-170Crossref PubMed Scopus (39) Google Scholar, 33Schafer Z.T. Parrish A.B. Wright K.M. Margolis S.S. Marks J.R. Deshmukh M. Kornbluth S. Cancer Res. 2006; 66: 2210-2218Crossref PubMed Scopus (49) Google Scholar). To directly examine this possibility, we determined the structural requirement for the apoptotic activity of PHAPI and provided strong evidence to support the model that the apoptotic activity of PHAPI is required for its tumor-suppressive function. Subcloning and Recombinant Protein Generation-Full-length PHAPI was cloned into pET-28a(+) vector (Novagen) as described previously (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). The DNA fragments encoding for individual deletion mutants of PHAPI were generated by PCR and subsequently cloned into BamHI/SalI sites of pET-28a(+) (Novagen) for expression of recombinant proteins. The nuclear localization defective mutant of PHAPI (NLDM) was generated by site-directed mutagenesis (residues 236–239, KRKR mutated to KLER). All plasmids were confirmed by DNA sequencing. Recombinant PHAPI and mutants were expressed as His6-tagged proteins in BL21(DE3) strain and purified using nitrilotriacetic acid-agarose (Qiagen) followed by Q-Sepharose chromatography. The purified proteins were dialyzed against Buffer A (20 mm HEPES, pH 7.5, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1.5 mm MgCl2, 1 mm dithiothreitol), and stored at -80 °C in small aliquots. PCR cloning was performed to generate plasmids for mammalian expression of differentially tagged PHAPI, including pFLAG-CMV-PHAPI (using BglII/SalI sites), pCDNA3.1-hygro-HA-PHAPI (BamHI/SalI), pCMV5-myc-PHAPI (BglII/SalI), and pEGFP-C1-PHAPI (BglII/SalI). To generate pWZL-hygro-PHAPI and its mutants for retroviral infection, the coding sequences were cut out by BamHI/SalI from the corresponding pcDNA3.1-hygro-HA constructs and then inserted into pWZL-hygro through the same sites. All plasmids were confirmed by DNA sequencing. Assay for PHAPI-stimulated Caspase Activity-The activity of PHAPI and its mutants to stimulate caspase activation was measured as described previously (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). This assay requires a Q-chromatographic fraction of HeLa cell extracts (HeLa S-100) that we named Q30. To prepare Q30, 10 ml of HeLa S-100 (∼60 mg of total protein) was loaded onto a 1-ml HiTrap Q column (Amersham Biosciences) pre-equilibrated with Buffer A. After sample loading, the column was washed with 10 ml of Buffer A. Subsequently, the column was eluted with Buffer A containing 300 mm NaCl, and the eluted protein peak (Q30, ∼4 ml) was collected. The Q30 fraction was stored at -80 °C in small aliquots. The in vitro assay for the apoptotic function of PHAPI and its truncation mutants was conducted in a 20-μl Buffer A system containing 3 μl of Q30, 100 nm cytochrome c, 10 μm dATP, 100 μm DEVD-aminomethylcoumarin substrate (Calbiochem), and 500 nm recombinant PHAPI or its truncation mutants as indicated. The reaction mixtures were incubated at 30 °C using a Xfluor4 spectrometry reader (TECAN, Inc.), and generation of fluorescent signal (relative fluorescent units) as a result of cleavage of DEVD-aminomethylcoumarin by caspase-3, was measured automatically every 10 min at wavelengths of 360/465 nm (excitation/emission). Alternatively, caspase-3 activity can also be detected using the in vitro translated, [35S]methionine-labeled caspase-3 as the substrate in a 60-min reaction. Light Scattering Analysis-Molar mass of purified Recombinant PHAPI was determined by static multiangle light scattering. Protein was injected onto a Superdex 200 HR 10/300 gel filtration column equilibrated in Buffer A. The chromatography system was coupled to a three-angle light scattering detector (mini-DAWN EOS) and refractive index detector (Optilab DSP, Wyatt Technology). The data were collected every 0.5 s at a flow rate of 0.25 ml/min. Data analysis was carried out using the ASTRA program (Wyatt Technology), yielding the molar mass and mass distribution (polydispersity) of the sample. Coimmunoprecipitation-HeLa cells were grown in 60-mm plates and maintained in Dulbecco's modified Eagle's medium containing 10% of fetal bovine serum. As indicated, plasmids encoding Myc-tagged PHAPI, FLAG-tagged PHAPI, or vector controls (1 μg each) were transiently transfected into HeLa cells with FuGENE 6 transfection reagent (Roche Applied Science). The cells were harvested 24 h after transfection. Cells were lysed by three cycles of freeze-and-thaw in Buffer L (20 mm HEPES, pH 7.5, 100 mm NaCl, 2 mm β-mercaptoethanol, and 0.2% Nonidet P-40) containing protease inhibitor mixture (Roche Applied Science). Subsequently the cell extracts were subjected to immunoprecipitation using protein-A-agarose coupled with 9E10 anti-Myc antibody for overnight. The immunoprecipitates and corresponding inputs were analyzed by Western blot against Myc, FLAG, and histone antibodies as indicated. PHAPI Translocation upon Stress Treatment-HeLa cells were grown in 60-mm plates and maintained in Dulbecco's modified Eagle's medium containing 10% of fetal bovine serum. One microgram of plasmid encoding GFP-conjugated PHAPI wild-type (WT), GFP-conjugated PHAPI NLDM mutant, or GFP alone was transiently transfected into HeLa cells with FuGENE 6 transfection reagent (Roche Applied Science). After 24 h of transfection, the cellular localization of the GFP proteins was detected by fluorescent microscopy. To test PHAPI translocation upon stress treatment, the HeLa cells expressing GFP-PHAPI (WT) were treated either with 0.25 μm staurosporine (Sigma) or 0.25 J/cm2 of UV light using a UV cross-linker (Stratagene). Pictures were taken at the indicated time points. Soft Agar Colony Formation Assay-Retroviruses were generated using pBABE-puro-Ras, pBABE-puro vector, pWZL-hygro-Myc, pWZL-hygro vector, pWZL-hygro-PHAPI, and its mutants as indicated. Primary mouse embryonic fibroblasts (cultured for three passages after isolation from mice) were plated in 10-cm plates (2 × 105/plate). After overnight incubation, they were infected by three rounds of infection (4 h for each round) with indicated retroviruses. Forty-eight hours after infection, cells were allowed to grow for 3 days in medium containing 2 μg/ml puromycin and 50 μg/ml hygromycin B. After recovering from drug selection for 12 h in antibiotics-free medium, viral-infected cells were trypsinized and plated for soft agar colony-formation assay. For each assay, 5 × 105 viral-infected cells were plated with soft agar medium to individual wells in 6-well plates. The plates were incubated for 2 weeks, and phase-contrast microscopic pictures were taken for each sample using a digital camera coupled to a microscope to show colony formation. Quantification and standard deviation were obtained from results of three independent experiments. Suspension Cell Culture and Measurement of Cellular Apoptosis-One million of each infected mouse embryonic fibroblasts as used in the soft agar colony formation assay were suspended in 2 ml of suspension medium (Dulbecco's modified Eagle's medium containing 1% of bovine serum albumin and 0.5% of methyl cellulose), and then seeded into the Ultra Low Cluster 6-Well Plate (Corning Inc.) and incubated in a 37 °C incubator with 5% CO2. Under this condition, cells are under suspension and anchorage-independent condition. Plates were periodically agitated to avoid cell aggregation. After 8 h of incubation, cells were harvested and lysed into Buffer A containing 140 mm additional KCl, 1% Nonidet P-40, and protease inhibitors. Caspase-3 activity in the cell extracts were measured to monitor suspension-triggered apoptosis in the cells expressing PHAPI or its individual mutants. The assay was conducted using 20 μg of individual cell extracts and 100 μm DEVD-aminomethylcoumarin substrate (Calbiochem) in 20-μl Buffer A containing 140 mm additional KCl. The reaction mixtures were incubated at 30 °C using a Xfluor4 spectrometry reader (TECAN, Inc.), and generation of fluorescent signal (relative fluorescent units) as a result of cleavage of DEVD-aminomethylcoumarin by caspase-3, was measured automatically every 10 min at wave-lengths of 360/465 nm (excitation/emission). Domain Analysis of PHAPI-We previously identified PHAPI/pp32 as a regulatory component of the mitochondria-mediated caspase activation pathway (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). PHAPI functions to enhance the activity of the apoptosome complex to activate caspase-9. However, PHAPI does not have any recognizable motif that can directly link to apoptosis. Therefore, to further understand the role of PHAPI in apoptosis, we sought to determine the domain structures of PHAPI required for its activity to stimulate caspase activation. PHAPI contains a short N-terminal fragment followed by four leucine-rich repeats (LRR1 to LRR4), and a short linker region (M region) connecting LRR4 to the highly negatively charged C terminus (Fig. 1A). We expressed and purified recombinant full-length PHAPI protein and a series of mutants with individual regions of the protein truncated (Fig. 1, A and B). Subsequently, we examined the activity of these recombinant proteins in stimulating Apaf-1/cytochrome c-mediated caspase activation. To do so, we used a chromatographic fraction (Q30) from HeLa cell cytosolic extracts and several other defined factors to reconstitute the caspase activation process (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). The Q30 fraction contains procaspase-9, procaspase-3, Apaf-1, and other required protein factors for the apoptotic activity of PHAPI (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar, 21Kim H.E. Jiang X. Du F. Wang X. Mol. Cell. 2008; 30: 239-247Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Caspase activation was triggered by addition of 100 nm purified cytochrome c and 10 μm nucleotide dATP and was measured by using cleavage of either a [35S]methionine-labeled procaspase-3 (Fig. 1C) or a fluorogenic peptide substrate of caspase-3 (Fig. 1D) as the readout. Based on both functional assays (Fig. 1, C and D), we found that there are two regions of PHAPI protein essential for its apoptotic activity. One is the acidic C-terminal fragment, and the other is the short M region immediately franking the C-terminal tail. LRR3 and LRR4 also appear to contribute to the apoptotic activity of PHAPI, because deletion of these two LRRs caused a modest but reproducible decrease of caspase activation measured by both assays (Fig. 1, C and D). Importantly, a truncated protein containing only the M region and the C-terminal tail of PHAPI, the two essential regions for the apoptotic activity of PHAPI, is sufficient to support the in vitro caspase stimulation activity of PHAPI (Fig. 1E). PHAPI but Not pp32R1 Can Stimulate Caspase Activation-PHAPI has a close homologous protein, pp32R1 (Fig. 2A). Intriguingly, instead of being a tumor suppressor like PHAPI, pp32R1 is an oncogene (29Brody J.R. Kadkol S.S. Mahmoud M.A. Rebel J.M. Pasternack G.R. J. Biol. Chem. 1999; 274: 20053-20055Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Kadkol S.S. Brody J.R. Pevsner J. Bai J. Pasternack G.R. Nat. Med. 1999; 5: 275-279Crossref PubMed Scopus (5) Google Scholar). We generated purified recombinant pp32R1 and examined its effect on Apaf-1-mediated caspase activation (Fig. 2B). Contrary to PHAPI, pp32R1 does not possess such apoptotic activity. This result, combined with the fact that PHAPI is a tumor suppressor, whereas pp32R1 is an oncoprotein, strongly suggests that the apoptotic activity and the tumor-suppressive capability of PHAPI have a causal relationship. Mechanistically, why is pp32R1 not able to stimulate caspase activation albeit being highly homologous to PHAPI? We reason that such functional difference might be related to the most diverse region of these two proteins, correlating to residues 140–163 of PHAPI (Fig. 2A). These residues are within the M region of PHAPI, which is essential for its apoptotic activity. To test this possibility, we generated a mutant form of PHAPI recombinant protein in which residues 140–163 were swapped with the correspondent residues of pp32R1 (we named this mutant as PRP, Fig. 2C). As expected, the PRP mutant of PHAPI indeed showed a much decreased activity to enhance caspase activation (Fig. 2D). Because pp32R1 is so similar to PHAPI but instead possesses oncogenic function, is it possible that pp32R1 promotes tumorigenesis by competing for the functional partner of PHAPI and thereby inhibiting its apoptotic activity? This turns out to not be the case, because in our in vitro caspase assay, neither pp32R1 nor the inactive PHAPI deletion mutants, ΔM and ΔC, can inhibit the activity of the full-length PHAPI to stimulate caspase activation (Fig. 3). There is another potential mechanism that might explain the oncogenic function of pp32R1. It was reported that PHAPI is a homo-trimeric protein based on its behavior in gel-filtration chromatography (34Ulitzur N. Rancano C. Pfeffer S.R. J. Biol. Chem. 1997; 272: 30577-30582Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We also observed that both native PHAPI purified from HeLa cell extracts and recombinant PHAPI were resolved in early fractions of gel-filtration chromatography, suggesting a large complex formed by homo-oligomerization (data not shown). If indeed PHAPI is a homo-trimer in cells and the homo-trimerization is required for its apoptotic activity, then one possibility for pp32R1 to abrogate this activity is to form inactive hetero-oligomers with PHAPI in cells. This potential activity would evade detection by our in vitro assay, because we used purified, recombinant PHAPI, which presumably was a stable homo-trimer before mixing with pp32R1. However, gel-filtration chromatography alone is not sufficient to determine whether PHAPI is an oligomer or not, because, in addition to molecular weight, other parameters such as the shape of a protein will also affect its gel-filtration chromatographic behavior. Therefore, we performed additional experiments to examine this possibility. Firstly, we performed coimmunoprecipitation experiment to examine whether differentially tagged PHAPI can interact with each other when co-transfected into HeLa cells. As shown in Fig. 4A, Myc-tagged PHAPI and FLAG-tagged PHAPI failed to interact with each other, whereas Myc-tagged PHAPI did co-precipitate endogenous histone, a previously reported interacting partner of PHAPI (25Seo S.B. Macfarlan T. McNamara P. Hong R. Mukai Y. Heo S. Chakravarti D. J. Biol. Chem. 2002; 277: 14005-14010Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This result argues against the possibility that PHAPI can form a homo-oligomer in cells. More conclusively, we measured the molecular mass of purified recombinant PHAPI by light scattering technique coupled with gel-filtration chromatography (Fig. 4B), the result (29.2 kDa) indicates that active PHAPI is a monomeric protein. Light scattering analysis also indicates that pp32R1 is a monomeric protein (data not shown). Therefore, our results suggest that pp32R1 exerts its oncogenic function through a mechanism not directly related to the apoptotic function of PHAPI. The Apoptotic Activity of PHAPI Contributes to Its Tumor-suppressive Function-PHAPI has been shown to be a tumor suppressor, but the underlying molecular basis was not determined. Our previous finding that PHAPI can enhance Apaf-1-mediated caspase activation provided a potential mechanistic explanation (20Jiang X. Kim H.E. Shu H. Zhao Y. Zhang H. Kofron J. Donnelly J. Burns D. Ng S.C. Rosenberg S. Wang X. Science. 2003; 299: 223-226Crossref PubMed Scopus (353) Google Scholar). The fact that the closely related pp32R1 is an oncoprotein and has no effect on caspase activation (Fig. 2) further supports this model. In addition, it was reported recently that, in certain human cancers, expression levels of PHAPI correlated with sensitivity of cancer cells to apoptosis (28Hoffarth S. Zitzer A. Wiewrodt R. Hahnel P.S. Beyer V. Kreft A. Biesterfeld S. Schuler M. Cell Death Differ. 2008; 15: 161-170Crossref PubMed Scopus (39) Google Scholar, 33Schafer Z.T. Parrish A.B. Wright K.M. Margolis S.S. Marks J.R. Deshmukh M. Kornbluth S. Cancer Res. 2006; 66: 2210-2218Crossref PubMed Scopus (49) Google Scholar). All these data are consistent with the possibility that the apoptotic activity of PHAPI is required for its tumor-suppressive function. To further test this hypothesis, we performed a soft agar colony formation assay to measure the tumor-suppressive function of a panel of PHAPI mutants with apoptotic activity defined in Fig. 1. With such an assay, we can determine whether the apoptotic activity of PHAPI
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