Regulation of Apoptosis and Cell Cycle Progression by MCL1статья из журнала
Аннотация: MCL1 (ML1 myeloid cellleukemia 1), a Bcl-2 (B-cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferatingcell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1Δ4A) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1Δ4A retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1Δ4A was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA. MCL1 (ML1 myeloid cellleukemia 1), a Bcl-2 (B-cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferatingcell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1Δ4A) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1Δ4A retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1Δ4A was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA. cyclin-dependent kinases proliferating cell nuclear antigen B-cell lymphoma-leukemia 2 myeloid cell leukemia 1 proline (P)-, glutamic acid (E)-, serine (S)-, and threonine (T)-rich polymerase chain reaction synthetic dropout 5-bromo-4-chloro-3-indeolyl β-d-galactopyranoside influenza hemagglutinin dithiothreitol enhanced green fluorescent protein 4,6-diamidino-2-phenylindole bromodeoxyuridine replication factor-C Apoptosis and cell cycle progression are closely linked processes under rigorous control. The integrated molecular mechanism to control apoptosis and cell cycle progression, namely the existence of regulatory molecule(s) that interface between apoptosis and cell cycle progression, has been implicated and extensively investigated. One such protein participating in the regulation of both apoptosis and cell cycle progression is p53, a tumor suppresser protein. Intriguingly, p53 transcriptionally activates both p21Waf1/Cip1, a cell cycle inhibitor (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar), and pro-apoptosis genes, such as bax (2Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (301) Google Scholar), noxa (3Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar),fas (4Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.W. Kruzel E. Radinsky R. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (687) Google Scholar), and p53-inducible genes (5Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2218) Google Scholar). The p21Waf1/Cip1 is a dual cell cycle inhibitor, functioning as an inhibitor of cyclin-dependent kinases (CDKs)1 and of proliferating cell nuclear antigen (PCNA) (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar). On the other hand, the activation ofbax, noxa, fas, and p53-inducible genes causes cells to undergo apoptosis (2Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 3Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar, 4Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.W. Kruzel E. Radinsky R. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (687) Google Scholar, 5Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2218) Google Scholar). Although the exact mechanism by which p53 preferentially activates genes related to either cell cycle progression or apoptosis induction is unclear, an emerging body of evidence suggests that the phosphorylation of p53 plays a critical role in the selective activation of certain genes by inducing the distinctive conformational change to and modifying the binding site preference of p53 (6Wang Y. Prives C. Nature. 1995; 376: 88-91Crossref PubMed Scopus (324) Google Scholar). Another example of molecules that participate both in apoptosis and cell cycle regulation is the E2F (7Field S. Tsai F. Kuo F. Zubiaga A. Kaelin Jr., W. Livingston D. Orkin S. Greenberg M. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar) family proteins. Transcription factors of the E2F family, composed of E2F-1-E2F-5, have been suggested to play a key role in the regulation of cell cycle progression (8Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4277) Google Scholar). Importantly, E2F transcriptionally activates both genes that regulate the S-phase entry, including c-myc (9Hiebert S.W. Lipp M. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3594-3598Crossref PubMed Scopus (222) Google Scholar), cyclin D (10Sala A. Nicolaides N.C. Engelhard A. Bellon T. Lawe D.C. Arnold A. Grana X. Giordano A. Calabretta B. Cancer Res. 1994; 54: 1402-1406PubMed Google Scholar), cyclin E (11DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (832) Google Scholar), and genes related to DNA synthesis, including dihydrofolate reductase (12Blake M.C. Azizkhan J.C. Mol. Cell. Biol. 1989; 9: 4994-5002Crossref PubMed Scopus (246) Google Scholar), thymidine kinase (13Dou Q.P. Markell P.J. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3256-3260Crossref PubMed Scopus (84) Google Scholar), and DNA polymerase α (10Sala A. Nicolaides N.C. Engelhard A. Bellon T. Lawe D.C. Arnold A. Grana X. Giordano A. Calabretta B. Cancer Res. 1994; 54: 1402-1406PubMed Google Scholar). In addition to its cell cycle regulatory function, E2F-1, one of the E2F family proteins, also functions as an apoptosis regulator. The overexpression of E2F-1 triggers apoptosis. The E2F-1−/− mice exhibit an excess of mature T cells due to a defect in thymocyte apoptosis (7Field S. Tsai F. Kuo F. Zubiaga A. Kaelin Jr., W. Livingston D. Orkin S. Greenberg M. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Taken together, E2F family proteins may function as both apoptosis and cell cycle regulators. Still another example of the integrated control of apoptosis and cell cycle progression is Survivin, one of the inhibitor of apoptosis protein family members (14Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (2981) Google Scholar). Originally, Survivin was found to prevent cells from undergoing apoptosis upon cytokine deprivation (14Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (2981) Google Scholar). Subsequently, it was shown that Survivin was highly up-regulated in the G2/M-phase of the cell cycle (15Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1710) Google Scholar). Survivin was found to be associated with microtubules and sustained cell survival during the G2/M-phase (15Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1710) Google Scholar). Thus, Survivin functions as a cell cycle regulatory protein and as an apoptosis inhibitor. The above evidence, along with other evidence, suggests that the molecules that regulate apoptosis can participate in the cell cycle regulation and vice versa. It is possible that proteins originally thought to regulate apoptosis may also have a role in cell cycle regulation. The B-cell lymphoma-leukemia 2 (Bcl-2) protein family represents one of the major groups of apoptosis regulatory proteins, sharing the same structural characteristics (16Cleary M.L. Smith S.D. Sklar J. Cell. 1986; 47: 19-28Abstract Full Text PDF PubMed Scopus (1068) Google Scholar, 17Chinnaiyan A.M. O'Rourke K. Lane B.R. Dixit V.M. Science. 1997; 275: 1122-1226Crossref PubMed Scopus (552) Google Scholar). At least 15 Bcl-2 family members have been identified in mammalian cells (18Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). Despite their structural similarities, Bcl-2 family members can either facilitate cell survival (pro-survival Bcl-2 subfamily) or promote cell death (pro-apoptosis Bax and Bcl-2 homologous 3 subfamilies) (18Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). MCL1 (myeloidcell leukemia 1) is a 37.3-kDa protein originally cloned from a differentiating myeloid leukemia 1 cell line (19Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar). Structurally and functionally, MCL1 belongs to the pro-survival Bcl-2 subfamily (20Reynolds J.E. Li J. Craig R.W. Eastman A. Exp. Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (128) Google Scholar) that also includes Bcl-xL, Bcl-2, and Bcl-w (18Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). However, MCL1 possesses two unique features that make it outstanding among the pro-survival Bcl-2 subfamily proteins. First, MCL1 is inducible upon proliferative (21Iyer V.R. Eisen M.B. Ross D.T. Schuler G. Moore T. Lee J.C.F. Trent J.M. Staudt L.M. Hudson Jr., J. Boguski M.S. Lashkari D. Shalon D. Botstein D. Brown P.O. Science. 1999; 283: 83-87Crossref PubMed Scopus (1707) Google Scholar) and differentiating (19Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar) stimuli. Second, the half-life of MCL1 is short (19Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar, 21Iyer V.R. Eisen M.B. Ross D.T. Schuler G. Moore T. Lee J.C.F. Trent J.M. Staudt L.M. Hudson Jr., J. Boguski M.S. Lashkari D. Shalon D. Botstein D. Brown P.O. Science. 1999; 283: 83-87Crossref PubMed Scopus (1707) Google Scholar) most likely because MCL1 contains two PEST sequences (22Rogers S. Wells R. Rechsteiner M. Science. 1986; 234: 364-368Crossref PubMed Scopus (1936) Google Scholar). These PEST sequences are not present in other Bcl-2 family proteins (19Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar). Interestingly, the PEST sequence is present in a number of cell cycle proteins, including cyclin D1, E (23Langenfeld J. Kiyokawa H. Sekula D. Boyle J. Dmitrovsky E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12070-12074Crossref PubMed Scopus (166) Google Scholar), G2 (24Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), F (25Bai C. Richman R. Elledge S.J. EMBO J. 1994; 13: 6087-6098Crossref PubMed Scopus (155) Google Scholar), I (26Nakamura T. Sanokawa R. Sasaki Y.F. Ayusawa D. Oishi M. Mori N. Exp. Cell Res. 1995; 221: 534-542Crossref PubMed Scopus (73) Google Scholar) and c-Fos (27Tsurumi C. Ishida N. Tamura T. Kakizuka A. Nishida E. Okumura E. Kishimoto T. Inagaki M. Okazaki K. Sagata N. Ichihara A. Tanaka K. Mol. Cell. Biol. 1995; 15: 5682-5687Crossref PubMed Scopus (127) Google Scholar). In order to investigate the mechanism of action and the potentially undiscovered functions of MCL1, we screened a human HeLa cell library with a yeast two-hybrid system using MCL1 as bait. Here we report a specific interaction between MCL1, an anti-apoptotic protein and a cell cycle regulatory protein, PCNA (28Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (704) Google Scholar). In this report, we propose that MCL1 is not only an anti-apoptotic protein but also a cell cycle regulator and that the cell cycle regulatory function of MCL1 is at least partially mediated through its interaction with PCNA. Transformed human embryonic kidney (293T) cells were purchased from the American Type Culture Collection (Manassas, VA). HeLa and U2OS cells (an osteosarcoma cell line) are generous gifts from Dr. Limin Gong (Institute of Molecular Medicine for Prevention of Human Diseases, Houston, TX). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, nonessential amino acids, and antibiotics (Life Technologies, Inc.). The cDNA fragments of MCL1, PCNA, Survivin, Bcl-xL, Bak, and Bax were obtained by a standard PCR technique (29Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K. (1998) in Current Protocols in Molecular Biology (Chanda, V. B., ed) unit 15.1, John Wiley & Sons, Inc., New York.Google Scholar) using appropriate primer sets. These cDNA fragments were then ligated in-frame to appropriate yeast and mammalian expression vectors. A mutant of MCL1, MCL1Δ4A, was generated by PCR-based strategies as described previously (30Gong L. Kamitani T. Fujise K. Caskey L.S. Yeh E.T. J. Biol. Chem. 1997; 272: 28198-28201Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In all cases, the authenticity of cloned constructs was confirmed by automated dideoxynucleotide sequencing (SeqWright Co., Houston, TX). The full-length MCL1 was cloned into pAS2.1 (CLONTECH, Palo Alto, CA), a vector that encodes GAL4 DNA-binding domain, and was used as bait.Saccharomyces cerevisiae PJ69-2A cells (MATa,CLONTECH) were transformed with pAS2.1-MCL1, using the lithium acetate method (31CLONTECHYeast Handbook. CLONTECH, Palo Alto, CA1997: 30-34Google Scholar). We then performed yeast mating between PJ69-2A cells containing pAS2.1-MCL1 and Y187 cells (MATα) containing a human HeLa cell library on pGAD GH (a vector that encodes GAL4 DNA-activating domain) for 27 h, according to the manufacturer's instructions (31CLONTECHYeast Handbook. CLONTECH, Palo Alto, CA1997: 30-34Google Scholar). Diploid yeast cells were selected for growth on synthetic dropout (SD) plates lacking adenine, histidine, leucine, and tryptophan (SD/−Ade/−His/−Leu/−Trp) for 14 days at 30 °C. Positive colonies were screened for β-galactosidase activity using a X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside) filter lift assay (31CLONTECHYeast Handbook. CLONTECH, Palo Alto, CA1997: 30-34Google Scholar). Plasmid DNAs were then isolated from colonies that activated all three yeast reporter genes (HIS3, ADE2, and lacZ) using the lyticase method (31CLONTECHYeast Handbook. CLONTECH, Palo Alto, CA1997: 30-34Google Scholar), propagated in the Escherichia coli, and analyzed by restriction digest and automated dideoxynucleotide sequencing (SeqWright). S. cerevisiae SFY526 cells (CLONTECH) were co-transformed with pAS2.1 containing full-length MCL1 or empty pAS2.1 and pGAD GH containing M01, the C-terminal half (137th to 261st amino acids) of PCNA or empty pGAD GH. Transformed cells were selected on SD/−Trp/−Leu plates for 7 days and subjected to the X-gal filter lift assay as described above. The blue color that developed within 8 h was considered to represent a positive interaction. Radiolabeled proteins for anin vitro binding assay were generated by a TNT Quick-coupled Transcription/Translation System (Promega, Madison, WI) according to the manufacturer's instruction, using [35S]methionine (Amersham Pharmacia Biotech) as a labeling agent. DNA templates were either circular plasmids or gel-purified PCR products, containing a T7 RNA polymerase promoter. The in vitro translated, influenza hemagglutinin-tagged PCNA (HA-PCNA) and another in vitrotranslated protein were added to Buffer A (50 mm HEPES, pH 7.5, 70 mm KCl, 0.5 mm ATP, 5 mmMgSO4, 1 mm dithiothreitol, 0.001% Nonidet P-40, 50 μm MG132, 2 μg/ml bovine serum albumin, aprotinin, phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma)) and allowed to form a complex at 4 °C for 90 min. HA-PCNA was then pulled down with rat anti-HA antibody (Clone 3F10, Roche Molecular Biochemicals) and sheep anti-rat polyclonal antibody conjugated to DynabeadsTM (M480, Dynal, Lake Success, NY). Immune complexes were then washed 5 times with Buffer A and once with Buffer B (Buffer A with 0.01% Nonidet P-40). Finally, precipitated proteins were eluted into SDS gel loading buffer (50 mmTris·Cl, pH 6.8, 100 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol), boiled for 5 min, subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by fluorography and a PhosphorImager system (Bio-Rad). The cDNA of MCL1 was cloned in-frame intoEcoRI and PstI sites of pEGFP-C2 (CLONTECH) to express MCL1 fused to the C terminus of an enhanced green fluorescent protein (EGFP). Approximately 1 × 106 293T cells were transfected with pEGFP-MCL1 or empty pEGFP using LipofectAMINE PLUS (Life Technologies, Inc.). Forty hours after the transfection, the cells were harvested by trypsinization and lysed by a nitrogen cavitation method (32Eichinger L. Bahler M. Dietz M. Eckerskorn C. Schleicher M. J. Biol. Chem. 1998; 273: 12952-12959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) (PARR Instrument Co, Moline, IL) in Buffer C (phosphate-buffered saline containing phenylmethylsulfonyl fluoride, aprotinin, and a mammalian cell protease inhibitor mixture (Sigma)). At least a pressure of 2000 pounds/square inch was applied. The disruption of both cell wall and nuclear membrane was confirmed under microscopy. The total cell lysate was cleared by centrifugation. An aliquot of approximately 2 × 105cells was incubated either with anti-PCNA monoclonal antibody (PC-10, IgG2, PharMingen, San Diego, CA) or with anti-GFP monoclonal antibody (Clone 3E6, IgG1, Quantum Biotechnology Inc., Montreal, Canada). Formed immune complexes were then precipitated by sheep anti-mouse antibodies conjugated to DynabeadsTM(M-280, Dynal). The precipitated complexes were washed 6 times with cold Buffer C, eluted into SDS gel loading buffer, boiled for 5 min, and subjected to a 12% SDS-PAGE. After the Western transfer, proteins on the nitrocellulose membrane were probed with anti-PCNA (PC-10) and anti-GFP (Clones 7.1 and 13.1, both IgG1, Roche Molecular Biochemicals) monoclonal antibodies. Bound antibodies were detected using subtype-specific rabbit anti-mouse IgG antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) conjugated to horseradish peroxidase with the ECLTM detection system (Amersham Pharmacia Biotech). Approximately 2 × 107 HeLa cells were harvested by trypsinization and suspended in Buffer A. Cells were then mechanically disrupted by the nitrogen cavitation with a pressure of 3000 pounds/square inch as described earlier. The cell lysate was cleared by centrifugation, and 500 μl of each was aliquoted into two tubes. Five micrograms of anti-PCNA monoclonal antibody (PC-10) were added to the first tube, and the same amount of control monoclonal antibody was added to the second tube. After incubation at 4 °C for 2 h, goat anti-mouse IgG conjugated to Dynabeads™ (M-280, Dynal) was added to the tubes, followed by an incubation of 1 h. The beads were washed six times with Buffer A. Immune complexes were eluted into SDS gel loading buffer, boiled for 5 min, separated on 12% SDS-PAGE in duplicate, transferred to nitrocellulose membranes, immunoprobed with anti-PCNA (PC-10) and anti-MCL1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) antibodies, and visualized by appropriate secondary antibodies conjugated to horseradish peroxidase (Southern Biotechnology Associates, Inc.) and the ECL detection system (Amersham Pharmacia Biotech). Immunofluorescence staining and confocal laser scanning microscopy was performed as described previously (33Liu J.L. Ye Y. Qian Z. Qian Y. Templeton D.J. Lee L.F. Kung H.J. J. Virol. 1999; 73: 4208-4219Crossref PubMed Google Scholar). In brief, U2OS cells were seeded in 4-well Lab-Teck™ glass chamber slides (Nalge Nunc International, Rochester, NY). The cells were fixed with fresh 4% paraformaldehyde in phosphate-buffered saline, briefly treated with methanol/acetone mixture (1:1 v/v) at −20 °C, blocked with 10% normal goat serum, and probed with rabbit anti-MCL1 polyclonal antibodies (Santa Cruz Biotechnology). Bound primary antibodies were detected by goat anti-rabbit antibody conjugated to Rhodamine Red X (Jackson ImmunoResearch Laboratories, West Grove, PA). Stained cells were analyzed with the Fluoview confocal laser scanning microscope (Olympus, Melville, NY), using the × 60 objective lens. The same analysis was performed to determine the intracellular localizations of overexpressed wild type and MCL1Δ4A, a mutant MCL1. The experiment was performed in duplicate. HeLa cells were seeded in 4-well Lab-Teck™ chamber slides, transfected either with pFLAG-MCL1, pFLAG-MCL1Δ4A, or pFLAG-LacZ using FuGENE6 (Roche Molecular Biochemicals), challenged with 5 μg/ml Etoposide for 12 h, and stained for the FLAG epitope using anti-FLAG antibody (M2, Sigma) and anti-mouse IgG conjugated to Rhodamine Red X (Jackson ImmunoResearch Laboratories). The nuclei were stained with DAPI (4,6-diamidino-2-phenylindole, Sigma). Cells were then examined under a Zeiss Axioskop fluorescent microscope (Carl Zeiss Ltd, Herts, UK), using appropriate filter sets. Cells that emitted red fluorescence were evaluated for their nuclear morphology. The condensed or fragmented nuclei were counted as apoptotic. An apoptotic index was then calculated as the number of red cells with apoptotic nuclear morphology divided by the number of total red cells counted. This experiment was performed in duplicate. Approximately 10,000 HeLa cells were seeded in 4-well Lab-Teck™ chamber slides (Nalge Nunc International). The next day, cells were transfected with pFLAG-LacZ, pFLAG-p21Waf1/Cip1, pFLAG-MCL1, or pFLAG-MCL1Δ4A using FuGENE6 (Roche Molecular Biochemicals) according to manufacturer's instruction. Cells were exposed to DNA-FuGENE6 complex for 5 h. Eighteen hours after the media change, cells were pulsed with 50 μm BrdUrd solution for 30 min at 37 °C. Harvested cells were first stained for incorporated BrdUrd, using a BrdUrd staining kit (Roche Molecular Biochemicals). In this procedure, bound anti-BrdUrd antibody was detected by goat anti-mouse Rhodamine Red X antibody (Jackson ImmunoResearch Laboratories). Cells were then stained for FLAG-tagged proteins with rabbit anti-FLAG antibody (Zymed Laboratories Inc., South San Francisco, CA) and goat anti-Rabbit-Cy2 antibody. The nuclei were counterstained with DAPI. The slides were examined under the Zeiss Axioskop fluorescent microscope (Carl Zeiss Ltd.), using appropriate filter sets. At least 150 cells (average = 364) were counted per chamber. The BrdUrd incorporation was defined as the number of green cells with red nuclei (FLAG- and BrdUrd-positive) divided by the number of green cells (FLAG-positive). Statistical analysis was performed using a generalized linear model with Duncan multiple comparison at the significance level of 0.05. In order to identify the protein(s) interacting with MCL1, we screened a human HeLa cell cDNA library using the yeast two-hybrid system. Among the 2 × 106 independent clones screened, one clone, named “M01,” not only grew on histidine-adenine dropout plates but also activated the β-galactosidase reporter gene by an X-gal colony lift assay. A DNA sequence analysis revealed that the clone M01 represents the C-terminal 124 amino acid residues of human PCNA, which is fused in frame to the GAL4 activation domain (Fig. 1 A). The presence of both MCL1 and M01 was necessary and sufficient for β-galactosidase reporter gene to be activated in yeast SFY526 cells (Fig. 1 A). Thus, we concluded that MCL1 specifically interacted with the C-terminal half of PCNA in the yeast two-hybrid system. We then performed an in vitro co-immunoprecipitation assay to test whether MCL1 would interact with the full-length PCNA. Here, we incubated in vitro translated, radiolabeled, influenza hemagglutinin (HA)-tagged PCNA with either in vitrotranslated, radiolabeled MCL1 or Survivin. Survivin is a member of inhibitor of apoptosis protein family, another anti-apoptotic protein family structurally unrelated to Bcl-2 family proteins (14Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (2981) Google Scholar, 15Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1710) Google Scholar). As is shown in Fig. 1 B, PCNA co-precipitated MCL1 (lanes 1 and 5) but not Survivin (lanes 2 and6). Moreover, MCL1 could not be precipitated in the absence of PCNA (lanes 3 and 7). Thus, MCL1 specifically interacted with full-length PCNA in vitro. We proceeded to test if MCL1 would interact with PCNA in mammalian cells in vivo. We transfected human embryonic kidney 293T cells with a plasmid encoding the cDNA of either the EGFP-tagged MCL1 or EGFP alone. As a result, EGFP-MCL1 and EGFP were found plentifully expressed in 293T cells (Fig. 1 C, lanes 1 and2, top panel). On the other hand, PCNA was found abundantly expressed in these cells without forced overexpression (Fig. 1 C, lanes 1 and 2, bottom panel). When we immunoprecipitated PCNA in the cell lysate by an anti-PCNA antibody (lanes 3 and 4, bottle panel), only EGFP-MCL1, not EGFP, was co-immunoprecipitated with PCNA (lanes 3 and 4, top panel). When we immunoprecipitated EGFP or EGFP-MCL1 in the cell lysate by anti-EGFP antibody (lanes 5and 6, top panel), only EGFP-MCL1, but not EGFP, co-immunoprecipitated PCNA (lanes 5 and 6,bottom panel). Thus, MCL1 specifically interacted with PCNA in mammalian cells in vivo. So far we have shown that MCL1 and PCNA interacted specifically with each other in overexpression systems (Fig. 1, A–C). We wished to evaluate whether this interaction could be demonstrated in a non-overexpression, native system. To this end, the lysate from 2 × 107 HeLa cells was incubated with anti-PCNA antibody or with control antibody at the same concentration. As is shown in Fig. 1 D, the lysate contained an equal amount of native MCL1 and PCNA as is shown in the bottom two panels (Total Cell Lysate (Input)). When the precipitated immune complexes were probed with anti-PCNA antibody, PCNA was found successfully precipitated by anti-PCNA antibody but not by the control antibody (Fig. 1 D, 2nd panel). When the immune complexes were probed with anti-MCL1 antibody, MCL1 was found co-precipitated with PCNA (Fig. 1 D, top panel, 1st column). There was no MCL1 precipitated in the absence of PCNA (Fig. 1 D,top panel, 2nd column). This result strongly suggests that MCL1-PCNA interaction is biologically significant since it can be demonstrated in native, non-overexpression system as well as an overexpression system. We then evaluated whether PCNA interacted with other Bcl-2 family proteins. By using the same system as the one described in Fig. 1 B, we tested the ability of PCNA to co-precipitate other pro- and anti-apoptotic Bcl-2 family proteins including Bcl-xL, Bak, or Bax. As is shown in Fig. 2, PCNA co-precipitated only MCL1 and not Bcl-xL, Bak, or Bax. Thus, MCL1 is an unusual Bcl-2 family protein that is capable of interacting with PCNA, a cell cycle regulatory protein.Figure 2MCL1 is unique among Bcl-2 family member proteins in its ability to interact with PCNA. The binding betweenin vitro translated, [35S]Met-labeled HA-PCNA and another in vitro translated, [35S]Met-labeled protein (i.e. MCL1, Bcl-xL, Bak, or Bax) was assessed in the system described in Fig. 1 B. Only MCL1 (lanes 1 and 6) and not Bcl-xL (lanes 2 and 7), Bak (lanes 3and 8), or Bax (lanes 4 and 9) was co-immunoprecipitated with PCNA in vitro.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Functionally, PCNA serves as a cofactor to DNA polymerase δ (34Bravo R. Frank R. Blundell P.A. Macdonald-Bravo H. Nature. 1987; 326: 515-5
Год издания: 2000
Авторы: Kenichi Fujise, Di Zhang, Juinn-Lin Liu, Edward T.H. Yeh
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Cell death mechanisms and regulation, Cancer-related Molecular Pathways, Retinoids in leukemia and cellular processes
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 275
Выпуск: 50
Страницы: 39458–39465