Collective Inhibition of pRB Family Proteins by Phosphorylation in Cells with p16INK4a Loss or Cyclin E Overexpression
2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês
10.1074/jbc.m007992200
ISSN1083-351X
AutoresSatoshi Ashizawa, Hiroko Nishizawa, Masafumi Yamada, Hideaki Higashi, Takuma Kondo, Heita Ozawa, Akira Kakita, Masanori Hatakeyama,
Tópico(s)RNA modifications and cancer
ResumoThe activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G1cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16INK4a-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of theRB gene. The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G1cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16INK4a-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of theRB gene. cyclin-dependent kinase hemagglutinin The retinoblastoma tumor suppressor protein pRB is a nuclear phosphoprotein that is ubiquitously expressed in somatic cells. It inhibits cell proliferation when ectopically expressed and is thought to play an important role in the growth decision-making in the late G1 phase of the cell cycle (1Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4986) Google Scholar, 2Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4326) Google Scholar). pRB is considered to inhibit cell proliferation by physically interacting with cellular proteins, most notably with the E2F family of transcription factors (3Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1100) Google Scholar, 4Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar, 5Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Upon complex formation with E2F, pRB inhibits transcriptional activation of those E2F-dependent genes whose products are essentially required for G1-to-S phase cell cycle transition. Furthermore, the pRB-E2F complex is capable of acting as a repressor against promoters containing E2F-binding sites, thereby actively repressing transcription in an E2F-dependent manner (4Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1981) Google Scholar, 5Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 6Weintraub S.J. Chow K.N. Luo R.X. Zhang S.H. He S. Dean D.C. Nature. 1995; 375: 812-815Crossref PubMed Scopus (459) Google Scholar). Sequential phosphorylation of pRB during G1by cyclin D-associated cyclin-dependent kinase (CDK)1-4/6 and cyclin E-CDK2 abolishes the ability of pRB to form physical complexes with cellular proteins, including E2F, and leads to G1-to-S phase cell cycle progression and subsequent cell division. The "p16INK4a-pRB" pathway is perturbed in most, if not all, cancer cells (7Delmer A. Tang R. Senamaud-Beaufort C. Paterlini P. Brechot C. Zittoun R. Leukemia ( Baltimore ). 1995; 9: 1240-1245PubMed Google Scholar, 8Ruas M. Brookes S. McDonald N.Q. Peters G. Oncogene. 1999; 18: 5423-5434Crossref PubMed Scopus (61) Google Scholar, 9Ruas M. Peters G. Biochim. Biophys. Acta. 1998; 1378: 115-177Crossref PubMed Scopus (877) Google Scholar, 10Russo A.A. Tong L. Lee J.O. Jeffrey P.D. Pavletich N.P. Nature. 1998; 395: 237-243Crossref PubMed Scopus (420) Google Scholar, 11Serrano M. Exp. Cell Res. 1997; 237: 7-13Crossref PubMed Scopus (279) Google Scholar, 12Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3392) Google Scholar). The changes include mutational inactivation of p16INK4a, cyclin D overexpression, and production of CDK4 mutants that cannot interact with p16INK4a. All of these changes lead to biochemical inactivation of pRB through phosphorylation, indicating that pRB plays a central role in preventing cellular transformation. On the other hand, genetic inactivation of a single copy of the RB gene predisposes only a limited set of malignancies such as retinoblastoma and osteosarcoma. This raises an intriguing possibility that dysregulation of the p16INK4a-pRB pathway gives rise to transformation in a broader range of cell types than those induced by RB gene inactivation. There are two proteins, p107 and p130, that share the so-called "pocket domain" with pRB (13Ewen M.E. Xing Y.G. Lawrence J.B. Livingston D.M. Cell. 1991; 66: 1155-1164Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 14Hannon G.J. Demetrick D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar, 15Li Y. Graham C. Lacy S. Duncan A.M. Whyte P. Genes Dev. 1993; 7: 2366-2377Crossref PubMed Scopus (302) Google Scholar, 16Mayol X. Grana X. Baldi A. Sang N. Hu Q. Giordano A. Oncogene. 1993; 8: 2561-2566PubMed Google Scholar). They are also capable of inhibiting cell growth upon ectopic expression (17Claudio P.P. Howard C.M. Baldi A. De Luca A. Fu Y. Condorelli G. Sun Y. Colburn N. Calabretta B. Giordano A. Cancer Res. 1994; 54: 5556-5560PubMed Google Scholar, 18Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 19Zhu L. Heuvel S.V.D. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar). Among the members of the pRB pocket protein family, p107 and p130 are more homologous to each other than to pRB and share unique properties allowing interaction with cyclins and CDKs (14Hannon G.J. Demetrick D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar, 15Li Y. Graham C. Lacy S. Duncan A.M. Whyte P. Genes Dev. 1993; 7: 2366-2377Crossref PubMed Scopus (302) Google Scholar, 20Cobrinik D. Whyte P. Peeper D.S. Jacks T. Weinberg R.A. Genes Dev. 1993; 7: 2392-2404Crossref PubMed Scopus (321) Google Scholar, 21Ewen M.E. Faha B. Harlow E. Livingston D.M. Science. 1992; 255: 85-87Crossref PubMed Scopus (164) Google Scholar, 22Faha B. Ewen M.E. Tsai L.H. Livingston D.M. Harlow E. Science. 1992; 255: 87-90Crossref PubMed Scopus (162) Google Scholar).Through the interaction, p107 or p130 is capable of inhibiting the kinase activity of cyclin-CDK (23Lacy S. Whyte P. Oncogene. 1997; 14: 2395-2406Crossref PubMed Scopus (57) Google Scholar, 24Zhu L. Harlow E. Dynlacht B.D. Genes Dev. 1995; 9: 1740-1752Crossref PubMed Scopus (234) Google Scholar). In addition, p107 and p130 selectively bind E2F-4 and E2F-5, whereas pRB preferentially binds E2F-1, E2F-2, and E2F-3 (25Beijersbergen R.L. Kerkhoven R.M. Zhu L. Carlee L. Voorhoeve P.M. Bernards R. Genes Dev. 1994; 8: 2680-2690Crossref PubMed Scopus (318) Google Scholar, 26Hijmans E.M. Voorhoeve P.M. Beijersbergen R.L. van't Veer L.J. Bernards R. Mol. Cell. Biol. 1995; 15: 3082-3089Crossref PubMed Scopus (207) Google Scholar, 27Ikeda M.A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3215-3220Crossref PubMed Scopus (225) Google Scholar, 28Moberg K. Starz M.A. Lees J.A. Mol. Cell. Biol. 1996; 16: 1436-1449Crossref PubMed Scopus (304) Google Scholar, 29Sardet C. Vidal M. Cobrinik D. Geng Y. Onufryk C. Chen A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2403-2407Crossref PubMed Scopus (302) Google Scholar, 30Vairo G. Livingston D.M. Ginsberg D. Genes Dev. 1995; 9: 869-881Crossref PubMed Scopus (289) Google Scholar). Hence, each member of the pRB family is likely to perform shared as well as unique cell cycle regulatory roles in a single cell. Furthermore, we (33Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 36Mori A. Higashi H. Hoshikawa Y. Imamura M. Asaka M. Hatakeyama M. Oncogene. 1999; 18: 6209-6221Crossref PubMed Scopus (14) Google Scholar) and others (30Vairo G. Livingston D.M. Ginsberg D. Genes Dev. 1995; 9: 869-881Crossref PubMed Scopus (289) Google Scholar, 31Chen P. Riley D.J. Chen Y. Lee W.H. Genes Dev. 1996; 10: 2794-2804Crossref PubMed Scopus (394) Google Scholar, 32Cobrinik D. Lee M.H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (378) Google Scholar, 34Hurford Jr., R.K. Cobrinik D. Lee M.H. Dyson N. Genes Dev. 1997; 11: 1447-1463Crossref PubMed Scopus (382) Google Scholar, 35Knudsen K.E. Weber E. Arden K.C. Cavenee W.K. Feramisco J.R. Knudsen E.S. Oncogene. 1999; 18: 5239-5245Crossref PubMed Scopus (56) Google Scholar) have recently shown that a different member of the pRB pocket protein family may become a key regulator of the cell cycle in different cell types. p107 and p130 are phosphorylated in a cell cycle-dependent manner. Like pRB, p107 can be a substrate for cyclin D-CDK4, and p107-induced cell cycle arrest was reportedly released by cyclin D-CDK4, but not by cyclin E-CDK2 (19Zhu L. Heuvel S.V.D. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar, 37Beijersbergen R.L. Carrel L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (236) Google Scholar, 38Xiao Z.X. Ginsberg D. Ewen M. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4633-4637Crossref PubMed Scopus (96) Google Scholar). In the case of p130, however, both D-type cyclins and cyclin E are capable of reversing the growth suppression mediated by p130. These observations raise the idea that the function of the entire pRB family of proteins is collectively regulated by phosphorylation, most likely through G1cyclin-CDK. Although this idea has long been suspected, there is little published information as to the functional regulation of p107 and p130 by phosphorylation. In this work, we generated p107 and p130 mutants that are resistant to cyclin-CDK-mediated phosphorylation. By expressing these phosphorylation-resistant molecules, we demonstrate that the growth-inhibitory activity as well as the cell differentiation/senescence-promoting activity of pRB, p107, and p130 are inactivated by phosphorylation. Our results indicate that elevation of the levels of pRB kinases through either p16INK4a loss or cyclin E overexpression abolishes the total activities of the pRB family proteins and, as a result, predisposes a broad spectrum of cells to a dysregulated cell cycle. The human osteosarcoma line SAOS-2 was provided by Dr. Phil Hinds (Harvard Medical School, Boston). The human osteosarcoma line U2-OS was obtained from American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium with 10% (for U2-OS) or 15% (for SAOS-2) fetal bovine serum. The phosphorylation-resistant pRB mutant, pRBΔS/T-P, was described previously (33Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar, 39Hamel P.A. Gill R.M. Phillips R.A. Gallie B.L. Oncogene. 1992; 7: 693-701PubMed Google Scholar). Other mutant constructs were generated by multiple rounds of oligonucleotide-mediated mutagenesis with the use of the Chameleon site-directed mutagenesis system (Stratagene) according to the manufacturer's instructions. cDNAs encoding p107N385, p107DE, and p107L19 were gifts from Dr. Liang Zhu (Albert Einstein College of Medicine, New York) (40Zhu L. Enders G. Lees J.A. Beijersbergen R.L. Bernards R. Harlow E. EMBO J. 1995; 14: 1904-1913Crossref PubMed Scopus (133) Google Scholar). The p130Δ846–1119 and p130Δ620–697 constructs were gifts of Dr. Peter Whyte (McMaster University, Ontario, Canada) (23Lacy S. Whyte P. Oncogene. 1997; 14: 2395-2406Crossref PubMed Scopus (57) Google Scholar). Some of the constructs were tagged with a hemagglutinin (HA) epitope at either the amino or carboxyl terminus. cDNAs encoding the wild-type and mutant pRB family proteins were inserted into mammalian expression vector pSP65-SRα (41Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). The pRc/CMV vector was used to express human cyclin E cDNA, and the pSV vector for adenovirus E1A (12 S and E1A-928 mutant). Either 20 μg of the indicated pocket protein expression plasmids or 10 μg of the pocket protein expression plasmids and 10 μg of E1A (either 12 S or E1A-928 mutant) expression plasmids were transfected into 1 × 106 U2-OS cells in a 100-mm plate by the calcium phosphate precipitation method as described previously (42Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar). After 2 days of culture in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, the transfected cells were harvested and lysed in E1A lysis buffer (42Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar). Cell lysates were first treated with anti-HA monoclonal antibody (12CA5) for 1 h, and the immune complexes were collected on protein A-Sepharose beads. After washing the beads, immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride filters (Millipore Corp.), and immunoblotted with appropriate antibodies. Proteins were visualized using the enhanced chemiluminescence detection system (ECL, PerkinElmer Life Sciences). The antibodies used were anti-cyclin A (H-432, sc-75, Santa Cruz Biotechnology), anti-E2F-4 (C-108, sc-512, Santa Cruz Biotechnology), and anti-adenovirus E1A (14161A, Pharmingen). 20 μg of the expression plasmid was transfected into U2-OS cells together with 2 μg of the puromycin resistance gene (pBabe-puro) (43Morgenstern L.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1903) Google Scholar) by the calcium phosphate precipitation method. At 16 h after the transfection, the cells were split into four 100-mm plates. The transfected cells were then selected in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in the presence of 2 μg/ml puromycin for 2 weeks. During the selection, the medium was changed twice a week. After the drug selection, the cells were stained by the May-Giemsa method, and the number of puromycin-resistant colonies was counted. The expression plasmid (20 μg) was transfected together with 2 μg of pBabe-puro into SAOS-2 cells by the calcium phosphate precipitation method. At 16 h after the transfection, the cells were split into four 100-mm plates, and the transfected cells were selected in Dulbecco's modified Eagle's medium with 15% fetal bovine serum in the presence of 0.5 μg/ml puromycin for 2 weeks. After the drug selection, the cells were stained with May-Giemsa solution and crystal violet. For cyclin E coexpression, 10 μg of the pRB family expression plasmid and 2 μg of pBabe-puro were transfected together with 10 μg of pCMV or pCMV-cyclin E. U2-OS cells were transfected with 20 μg of the pRB family expression plasmid together with 2 μg of the CD20 expression plasmid (pCMV-CD20) (40Zhu L. Enders G. Lees J.A. Beijersbergen R.L. Bernards R. Harlow E. EMBO J. 1995; 14: 1904-1913Crossref PubMed Scopus (133) Google Scholar) as a marker. 40 h after the transfection, cells were harvested and treated with an anti-CD20 antibody (fluorescein isothiocyanate-labeled CD20, Becton Dickinson) for 20 min on ice, washed with phosphate-buffered saline, and fixed in 70% ethanol on ice. The cells were washed again and resuspended in 100 μl of phosphate-buffered saline containing 25 μg/ml RNase A for 20 min. Prior to flow cytometry, 700 μl of propidium iodide solution (100 μg/ml propidium iodide and 0.1% sodium citrate) was added to the cell suspension, and cells were incubated for another 15 min on ice. The intensity of propidium iodide staining was measured by a FACSCalibur (Becton Dickinson) on cell populations that were positive for CD20 expression to determine the DNA content. Cell cycle profiles of the CD20-positive cells were analyzed using CELL Quest and ModFit cell cycle analysis software (Becton Dickinson). To address the role of cyclin-associated CDK-dependent phosphorylation in the function of p107 and p130, we generated phosphorylation-resistant mutants as described previously in a study on the function of pRB (33Hoshikawa Y. Mori A. Amimoto K. Iwabe K. Hatakeyama M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8574-8579Crossref PubMed Scopus (35) Google Scholar). Human p107 possesses 18 Ser-Pro or Thr-Pro ((Ser/Thr)-Pro) motifs that are potential phosphorylation targets for cyclin-CDK. The serine and threonine residues making up all of the (Ser/Thr)-Pro motifs were replaced by alanine to generate p107ΔS/T-P. Similarly, all of the 27 (Ser/Thr)-Pro motifs in human p130 were mutated to generate p130ΔS/T-P. In the case of p130, p130ΔCDK was also made by mutating 12 serine/threonine residues constituting the canonical CDK consensus motif ((S/T)PX(L/R)) to alanine residues. All of the pocket protein mutants used in this work are summarized in Fig. 1. Since the phosphorylation-resistant mutants possess multiple point mutations, we first investigated whether or not these proteins retain sufficient structural integrity to interact with cyclins and E2F proteins in the manner of their respective wild-type proteins. To do so, cDNA encoding the wild-type or the phosphorylation-resistant mutant protein was transfected into U2-OS cells. As demonstrated in Fig. 2(A, upper panel, lanes 2,4, and 6; and C, upper panel, lanes 2, 4, 6,10, 12, and 14), the wild-type pocket proteins were detected as broad bands, indicating that they were variably phosphorylated. In contrast, the phosphorylation-resistant mutants were all detected as fast migrating bands (Fig. 2,A, upper panel, lanes 3, 5,7, and 8; and C, upper panel, lanes 3, 5, 7,8, 11, 13, 15, and16), indicating that they were resistant to phosphorylation in cells. In sequential immunoprecipitation-immunoblotting analyses, endogenous E2F-4 proteins were co-immunoprecipitated with all wild-type and mutant pocket proteins, with the exception of p130ΔS/T-P (Fig. 2,A, middle panel; and B). In each case, E2F-4 molecules coprecipitated with the mutant pocket proteins were detected in amounts similar to those coprecipitated with their respective wild-type counterparts, although the expression levels of the mutant proteins were significantly less than those of the wild-type proteins. This indicates at least that the affinities of the mutant pocket proteins for binding E2F-4 are not reduced despite introduction of multiple mutations. Notably, the E2F-4-binding affinity of pRB or pRBΔS/T-P was significantly less than that of p107 or p107ΔS/T-P (Fig. 2, A and B) as reported previously (18Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 44Ginsberg D. Vairo G. Chittenden T. Xiao Z. Xu G. Wydner K.L. DeCaprio K.L. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 2665-2679Crossref PubMed Scopus (301) Google Scholar). Furthermore, wild-type p107, wild-type p130, p107ΔS/T-P, and p130ΔCDK, but not p130ΔS/T-P, physically interacted with endogenous cyclin A (Fig. 2 A, lower panel). Again, in each case, coprecipitated cyclin A levels were found to be similar to the levels coprecipitated with their respective wild-type counterparts. In contrast, wild-type pRB and pRBΔS/T-P did not associate with cyclin A because they do not possess cyclin A-binding spacer sequences. These observations indicate that p107ΔS/T-P, p130ΔCDK, and pRBΔS/T-P are capable of interacting with cellular targets with affinities comparable to those of their respective wild-type molecules. Conformation of the phosphorylation-resistant mutant as the "pocket protein" was further examined with the use of the adenovirus E1A 12 S product, which specifically binds the pocket domains and inactivates the pRB family proteins (45Mayol X. Garriga J. Grana X. Oncogene. 1996; 13: 237-246PubMed Google Scholar, 46Whyte P. Williamson N.M. Harlow E. Cell. 1989; 56: 67-75Abstract Full Text PDF PubMed Scopus (542) Google Scholar, 47Hu Q. Dyson N. Harlow E. EMBO J. 1990; 9: 1147-1155Crossref PubMed Scopus (247) Google Scholar). Upon transient coexpression in U2-OS cells, the mutant pRB family molecules, except p130ΔS/T-P, formed stable complexes with E1A to levels comparable to those associated with their respective wild-type counterparts (Fig. 2 C). Moreover, an E1A point mutant, E1A-928, which does not bind to wild-type pRB, did not bind pRBΔS/T-P as well (Fig. 2 C, middle panel, lanes 10 and 11). The E1A mutant is also known to bind p107 and p130 less effectively. Consistently, p107ΔS/T-P and p130ΔCDK exhibited reduced affinities for E1A-928, as is the case with wild-type p107 and p130 (Fig. 2 C,middle panel, compare lanes 4–7 with lanes 12–15) (48Moran E. Zerler B. Harrison T.M. Mathews M.B. Mol. Cell. Biol. 1986; 6: 3470-3480Crossref PubMed Scopus (137) Google Scholar, 49Wang H.H. Rikitake Y. Carter M.C. Yaciuk P. Abraham S.E. Zerler B. Moran E. J. Virol. 1993; 67: 476-488Crossref PubMed Google Scholar, 50Parreno M. Garriga J. Limon A. Mayol X. Beck Jr., G.R. Moran E. Grana X. J. Virol. 2000; 74: 3166-3176Crossref PubMed Scopus (14) Google Scholar). These observations provide further evidence that the structural conformations of the phosphorylation-resistant pocket protein mutants pRBΔS/T-P, p107ΔS/T-P, and p130ΔCDK are indistinguishable from those of their respective wild-type molecules, pointing to the notion that these mutants function through normal biological pathways that involve their wild-type counterparts when expressed in cells. On the other hand, p130ΔS/T-P, which has 27 point mutations, lost its structural integrity as a pocket protein because of the introduced mutations. It has been shown that U2-OS cells are highly resistant to pRB overexpression (19Zhu L. Heuvel S.V.D. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar). Since the U2-OS cells do not express functional p16INK4a (51Suzuki T.I. Higashi H. Yoshida E. Nishimura S. Kitagawa M. Biochem. Biophys. Res. Commun. 1997; 234: 386-392Crossref PubMed Scopus (9) Google Scholar), a specific inhibitor of cyclin D-CDK4/6, the cells exhibit dysregulated pRB kinase activity (52Hofmann F. Martelli F. Livingston D.M. Wang Z. Genes Dev. 1996; 10: 2949-2959Crossref PubMed Scopus (214) Google Scholar). It is therefore considered that, in U2-OS cells, the dysregulated pRB kinase instantly inactivates ectopically expressed wild-type pRB proteins. If this is the case, then the growth of U2-OS cells must be inhibited by phosphorylation-resistant pRB. To address this, a cDNA expression vector for wild-type pRB or the phosphorylation-resistant pRB mutant, pRBΔS/T-P, was cotransfected with the CD20 expression vector, and the cell cycle distribution of the CD20-positive cells at 40 h after the transfection was examined with the use of a flow cytometer. As previously reported (19Zhu L. Heuvel S.V.D. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar), ectopic expression of pRB had very little effect on the cell cycle distribution profile of U2-OS cells (Fig. 3). On the other hand, pRBΔS/T-P caused severe G1 cell cycle arrest in U2-OS cells (Fig. 3). This indicates that, in these cells, the growth-suppressive activity of pRB is neutralized by phosphorylation, most likely through the dysregulated cyclin D-CDK kinase because of the lack of p16INK4a. The U2-OS cells allowed us to examine whether the growth-suppressive activity of p107 or p130 is also under the control of cyclin-CDK-mediated phosphorylation. To this end, we transiently introduced wild-type p107 or p107ΔS/T-P together with CD20 into U2-OS cells (Fig. 3). Whereas the expression of wild-type p107 did not affect the cell cycle distribution significantly, phosphorylation-resistant p107 provoked strong accumulation of cells in G1 phase, indicating that the mutant molecules caused G1 cell cycle arrest in U2-OS cells. This indicates that p107 is capable of inhibiting cell cycle progression and that the activity is under phosphorylation control in U2-OS cells (Fig. 3). The wild-type and two forms of phosphorylation-resistant p130 molecules, p130ΔS/T-P and p130ΔCDK, were next examined for their growth regulatory activity in U2-OS cells. In contrast to pRB and p107, p130, even in its phosphorylation-resistant forms, did not significantly affect the cell cycle profile of U2-OS cells in this transient expression assay (Fig. 3). Given the observation that p130ΔS/T-P cannot bind E2F or cyclin A (Fig. 2), this finding is consistent with the conclusion that the mutant is functionally inactive. In contrast, since p130ΔCDK can still bind with the target molecules, the growth-inhibitory activity of p130, if it exists, may be significantly weaker than that of pRB or p107 in U2-OS cells. We next investigated the long-term effects of the pRB family proteins on the growth of U2-OS cells. To do so, we transfected cDNA expression vectors for the pRB family proteins together with the puromycin resistance gene. After selection of the transfected cells with puromycin, the number of puromycin-resistant colonies was counted. As shown in Fig. 4, expression of wild-type pocket proteins in U2-OS cells resulted in a weak reduction of puromycin-resistant colonies, indicating that these proteins are growth-inhibitory. As expected from the transient assay, the phosphorylation-resistant pRB and p107 molecules reduced puromycin-resistant colonies quite strongly (Fig. 4). Furthermore, in this colony formation assay, the phosphorylation-resistant p130 mutant p130ΔCDK suppressed U2-OS colony formation more effectively than wild-type p130. This indicates that p130 is also capable of inhibiting cell proliferation and that this growth-inhibitory activity is under phosphorylation control. In contrast, p130ΔS/T-P, which lacks all 27 (Ser/Thr)-Pro motifs, was not growth-suppressive in the colony assay (Fig. 4). Accordingly, as suggested from its inability to bind E2F-4 or cyclin A, the introduction of mutations into one or several (Ser/Thr)-Pro sites present in p130ΔCDK but absent in p130ΔS/T-P appears to provoke structural inactivation of p130. Alternatively, since p130 receives multiple phosphorylation and its function is reportedly modified depending on phosphorylation status (53Mayol X. Garriga J. Grana X. Oncogene. 1995; 11: 801-808PubMed Google Scholar), a certain degree of basal phosphorylation might be required for the activation of p130 as a growth suppressor. If this is the case, then p130ΔS/T-P may be functionally inactive because it cannot receive activating phosphorylation by the proline-directed kinases that target the (Ser/Thr)-Pro motifs. In any case, the failure of p130ΔS/T-P to inhibit cell growth argues against the idea that cell cycle-inhibitory effects of the phosphorylation-resistant mutants are due to the overexpression and accumulation of functionally inactive pocket proteins. Expression of pRB in pRB-defective SAOS-2 human osteosarcoma cells causes strong G1 cell cycle arrest that is associated with a large flat cell formation (42Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar, 54Huang H.J. Yee J.K. Shew J.Y. Chen P.L. Bookstein R. Friedmann T. Lee E.Y. Lee W.H. Science. 1988; 242: 1563-1566Crossref PubMed Scopus (627) Google Scholar, 55Qin X.Q. Chittenden T. Livingston D.M. Kaelin Jr., W.G. Genes Dev. 1992; 6: 953-964Crossref PubMed Scopus (358) Google Scholar, 56Templeton D.J. Park S.H. Lanier L. Weinberg R.A. Proc. Natl. Acad. Sc
Referência(s)