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• W1982074920 abstract "GSK-3β-dependent phosphorylation of cyclin D1 at a conserved C-terminal residue, Thr-286, promotes CRM1-dependent cyclin D1 nuclear export. Herein, we have identified a short stretch of residues adjacent to Thr-286 that mediates CRM1 association and thus cyclin D1 nuclear export. We found that disruption of this hydrophobic patch, stretching from amino acids 290 to 295 within cyclin D1, results in constitutively nuclear cyclin D1-CDK4 complexes with an increased propensity to potentiate transformation of murine fibroblasts. Our data support a model wherein deregulation of cyclin D1 nuclear export might contribute to human neoplastic growth. GSK-3β-dependent phosphorylation of cyclin D1 at a conserved C-terminal residue, Thr-286, promotes CRM1-dependent cyclin D1 nuclear export. Herein, we have identified a short stretch of residues adjacent to Thr-286 that mediates CRM1 association and thus cyclin D1 nuclear export. We found that disruption of this hydrophobic patch, stretching from amino acids 290 to 295 within cyclin D1, results in constitutively nuclear cyclin D1-CDK4 complexes with an increased propensity to potentiate transformation of murine fibroblasts. Our data support a model wherein deregulation of cyclin D1 nuclear export might contribute to human neoplastic growth. Progression through G1 phase is initiated by mitogenic stimulation, which in turn initiates the expression and assembly of the D-type cyclins (D1, D2, D3) with their catalytic partner CDK4/6. 1The abbreviations used are: CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; GSK-3β, glycogen synthase kinase 3β; HA, hemagglutinin; FCS, fetal calf serum. The cyclin D/CDK4 kinase has two functions necessary for cell cycle progression: phosphorylation-dependent inactivation of the retinoblastoma protein family members such as pRb, p107, and p130 (1Harbour J.W. Luo R.X. Dei Santi A. Postigo A.A. Dean D.C. Cell. 1999; 98: 859-869Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar, 2Calbo J. Parreno M. Sotillo E. Yong T. Mazo A. Garriga J. Grana X. J. Biol. Chem. 2002; 277: 50263-50274Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 3Leng X. Noble M. Adams P.D. Qin J. Harper J.W. Mol. Cell. Biol. 2002; 22: 2242-2254Crossref PubMed Scopus (73) Google Scholar, 4Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (222) Google Scholar, 5Farkas T. Hansen K. Holm K. Lukas J. Bartek J. J. Biol. Chem. 2002; 277: 26741-26752Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and the stoichiometric association with Cip/Kip family proteins (6Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3179) Google Scholar, 7Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar, 8LaBaer J. Garrett M.D. Stevenson L.F. Slingerland J.M. Sandhu C. Chou H.S. Fattaey A. Harlow E. Genes Dev. 1997; 11: 847-862Crossref PubMed Scopus (1225) Google Scholar, 9Alt J.R. Gladden A.B. Diehl J.A. J. Biol. Chem. 2002; 277: 8517-8523Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). This association facilitates cyclin D1/CDK4 activity through increased nuclear retention and subunit assembly while simultaneously preventing Cip/Kip access to cyclin E-CDK2 complexes, which would result in the inhibition of the cyclin E/CDK2 kinase (9Alt J.R. Gladden A.B. Diehl J.A. J. Biol. Chem. 2002; 277: 8517-8523Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 10Cheng M. Sexl V. Sherr C.J. Roussel M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1091-1096Crossref PubMed Scopus (471) Google Scholar, 11Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (974) Google Scholar, 12Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar, 13Muraoka R.S. Lenferink A.E. Law B. Hamilton E. Brantley D.M. Roebuck L.R. Arteaga C.L. Mol. Cell. Biol. 2002; 22: 2204-2219Crossref PubMed Scopus (105) Google Scholar, 14Muraoka R.S. Lenferink A.E. Simpson J. Brantley D.M. Roebuck L.R. Yakes F.M. Arteaga C.L. J. Cell Biol. 2001; 153: 917-932Crossref PubMed Scopus (64) Google Scholar). Although cyclin D1 accumulates in the nucleus during the G1 interval, it relocalizes to the cytoplasm during S phase. The essential functions of cyclin D1 require its nuclear localization, and thus the redistribution of cyclin D1 complexes to the cytoplasm following G1 implies that regulation of cyclin D1 nucleocytoplasmic distribution is necessary for maintaining cellular homeostasis. These alterations in the subcellular distribution of cyclin D1 during the cell cycle are subject to mitogenic regulation (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar, 16Baldin V. Lukas J. Marcote M.J. Pagano M. Draetta G. Genes Dev. 1993; 7: 812-821Crossref PubMed Scopus (1438) Google Scholar). It is now clear that phosphorylation of cyclin D1 at Thr-286 by GSK-3β promotes CRM1 binding, which then shuttles cyclin D1 to the cytoplasm (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar) for subsequent degradation via the 26S proteasome (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). Although these data imply that phosphorylation of Thr-286 enables CRM1 binding, the residues within cyclin D1 that direct CRM1 binding have not been elucidated. Although cyclin D1 is overexpressed in a number of malignancies, it is clear that overexpression of the wild type protein is not by itself sufficient to induce a transformed cellular phenotype (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar, 18Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.Y. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar). In contrast, we found previously that neither the cyclin D1 mutant, cyclin D1-T286A, nor an alternatively spliced cyclin D1 variant lacking the fifth exon can be phosphorylated by GSK-3β, and thus both are refractory to phosphorylation-dependent nuclear export and are capable of driving transformation of murine fibroblasts in the absence of a collaborating oncogene (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar, 20Lu F. Gladden A.B. Diehl J.A. Cancer Res. 2003; 63: 7056-7061PubMed Google Scholar). This suggests that deregulation of cyclin D1 nuclear export results in increased cyclin D1 oncogenic capacity. Herein we describe the identification of residues within cyclin D1 that direct CRM1 association. Our results demonstrate that hydrophobic residues adjacent to Thr-286 and within the C-terminal nine amino acids of cyclin D1 mediate CRM1 binding. Cell Culture Conditions and Transfections—NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium containing glutamine supplemented with antibiotics (Cellgro) and 10% FCS (Gemini). Procedures for manipulation of baculoviruses were described previously (21Summers M.D. Smith G.E. Tex. Agric. Exp. St. Bull. 1987; : 1555Google Scholar). Site-directed mutagenesis of D1-V290/295A mutant was achieved by using QuikChange (Stratagene), substituting valines 290 and 293 and isoleucine 295 to alanines with the following primers: forward 5′-TGCACGCCCACCGACGTGCGAGATGTGGACATC TGAGGGCCACCG-3′ and reverse 5′-CGGTGGCCCTCAGGCGTCCGCATCTCG CGCGTCGGTGGGCGT-3′. The derivation of NIH-3T3 cells overexpressing FLAG-tagged cyclin D1 and D1 mutants was as described previously (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar). Cell lines were maintained on a passage protocol wherein 9 × 105 cells were passaged per 60-mm dish every third day (22Todaro G.J. Green H. J. Cell. Biol. 1963; 17: 299-313Crossref PubMed Scopus (2011) Google Scholar). Transient expression of HA-tagged GSK-3β or HA-tagged CRM1 was performed as described previously (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). Immunoblotting—For Western analysis, cells were lysed in EBC buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 20 units/ml aprotinin, 5 μg/ml leupeptin, 0.4 mm NaVO4, 0.4 mm NaF). Total cellular proteins (200 μg) were resolved on denaturing polyacrylamide gels, electrophoretically transferred to nitrocellulose membranes (MSI, Westborough, MA), and blotted with the cyclin D1 monoclonal antibody (D1-17-13G). Detection of GSK-3β was performed as described previously (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). Expression and Purification of Proteins—FLAG-D1 mutants were cloned into the pVDL-1393 baculoviral expression vector (Pharmingen), and virus was isolated according to established procedures as described previously (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). After infection of insect Sf9 cells at high multiplicity, cells were lysed in EBC buffer and clarified by sedimentation in a microcentrifuge for 10 min. Recombinant proteins were then subjected to precipitation with epitope-specific antibodies and detected by immunoblot. Immunofluorescence—NIH-3T3 cells seeded on glass coverslips were transfected with expression vectors encoding the indicated cDNAs. Cells were fixed at 48 h after transfection using methanol-acetone (1:1). For visualization of cyclin D1, coverslips were stained with a mouse-specific cyclin D1 monoclonal antibody (D1-17-13G) in phosphate-buffered saline containing 10% FCS. Secondary antibody staining was performed for 30 min using fluorescein isothiocyanate-conjugated anti-mouse (Amersham Biosciences). DNA was visualized using Hoescht 33258 dye at a 1:500 dilution. Coverslips were mounted on glass slides with Vectashield (Vector Laboratories). Protein Turnover Analysis—Equivalent numbers of NIH-3T3 cells overexpressing either FLAG-tagged wild type or mutant cyclin D1 were seeded in 10-cm dishes. The following day, cells were treated with the protein synthesis inhibitor cycloheximide (100 μg/ml, Sigma), incubated for the indicated times, and harvested in SDS-sample buffer. Cyclin D1 was detected by Western blot analysis. Transformation Assays—NIH-3T3 cells and derivatives overexpressing the indicated cyclin D1 constructs were plated at 1.5 × 105 cells/well of a 6-well dish. Cells were cultured in medium containing 5% FCS. Foci were visualized after 21-28 days with Wright Giemsa stain (Sigma). Anchorage-independent growth of NIH-3T3, D1-3T3, D1-T286A-3T3, and D1-V290/295A was performed as described previously (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar). Cyclin D1 Residues 290-295 Mediate CRM1 Binding— CRM1 directs nuclear export of target proteins via direct binding to leucine/hydrophobic stretches of amino acids referred to as a nuclear export signal (23Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (921) Google Scholar). Because phosphorylation of Thr-286 is required for CRM1 association with cyclin D1, we reasoned that phosphorylation of this residue might result in a conformational change that allows CRM1 access to a region of cyclin D1 adjacent to this site of phosphorylation. A stretch of 10 highly conserved amino acids exists within the C terminus of cyclins D1 and D2 (286TPT-DVRDVD(I/L) in cyclin D1) (24Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (990) Google Scholar) with the hydrophobic residues (in boldface) being conserved in cyclin D3 as well (Fig. 1A). Since both the spacing of these residues closely resembles that of other known CRM1 substrates such as cyclin B1 (25Toyoshima F. Moriguchi T. Wada A. Fukuda M. Nishida E. EMBO J. 1998; 17: 2728-2735Crossref PubMed Scopus (281) Google Scholar, 26Yang J. Bardes E.S. Moore J.D. Brennan J. Powers M.A. Kornbluth S. Genes Dev. 1998; 12: 2131-2143Crossref PubMed Scopus (283) Google Scholar) and a high degree of homology within this region is retained among all three D-type cyclins, we sought to evaluate the potential of these residues to mediate the cyclin D1-CRM1 association. We used site-directed mutagenesis to engineer a mutant D1-V290/295A, wherein the two valines and the isoleucine residue within residues 290-295 of cyclin D1 (VRDVDI) were mutated to alanine (ARDADA). Alanine substitutions have previously been shown to abrogate CRM1 association (27Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (988) Google Scholar, 28Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (1006) Google Scholar). We first determined whether alanine substitutions abrogated cyclin D1-CRM1 binding. NIH-3T3 cells were transfected with HA-tagged CRM1 along with either FLAG-D1 as a positive control, FLAG-D1-T286A as a negative control, or FLAG-D1-V290/295A. Lysates prepared from these cells were analyzed by immunoblotting or subjected to precipitation with the 12CA5 monoclonal antibody, which recognizes the HA epitope, followed by immunoblotting with antibodies directed toward either the HA epitope or cyclin D1. Although wild type cyclin D1 co-precipitated with CRM1, consistent with previous analysis (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar, 29Connor M.K. Kotchetkov R. Cariou S. Resch A. Lupetti R. Beniston R.G. Melchior F. Hengst L. Slingerland J.M. Mol. Biol. Cell. 2003; 14: 201-213Crossref PubMed Scopus (161) Google Scholar), neither D1-T286A nor D1-V290/295A was detected in the CRM1 precipitates (Fig. 1B). The inability of D1-V290/295A to associate with CRM1 was not due to reduced expression of this protein (lanes 4-6), suggesting that alanine substitutions within this region disrupt CRM1-D1 interaction. Overexpression of CRM1 can drive wild type cyclin D1 into the cytoplasm, whereas the phosphorylation-deficient D1-T286A mutant is refractory to the enforced nuclear export driven by CRM1 overexpression (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar). It stands to reason that cyclin D1 mutants deficient in CRM1 binding, such as D1-V290/295A, should be refractory to CRM1-directed nuclear export and thus constitutively nuclear. To address this possibility, we examined the subcellular localization of D1-V290/295A in asynchronously proliferating cells. The localization of cyclin D1 varied, with ∼50% of the cells exhibiting primarily nuclear localization, whereas in the remaining cells it localized either to the cytoplasm or to both the nucleus and the cytoplasm (Fig. 1, C and D). This distribution of nuclear cyclin D1 approximates the percentage of cells in G1 phase in an asynchronous population (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). In contrast, D1-V290/295A was nuclear in more than 90% of the cells (Fig. 1, C and D). To assess the capacity of CRM1 to regulate localization, we determined whether ectopic CRM1 could drive wild type cyclin D1 or D1-V290/295A into the cytoplasm. Whereas expression of CRM1 shuttled wild type cyclin D1 out of the nucleus (Fig. 1, C and D (quantitation)), D1-V290/295A remained nuclear in the presence or absence of ectopic CRM1 (1, C and D (quantitation)). Although these results suggest that this region of cyclin D1 mediates CRM1 binding, its close proximity to Thr-286 could disrupt GSK-3β-mediated phosphorylation and thereby indirectly disrupt CRM1 association. To examine the phosphorylation state of D1-V290/295A, we performed immunoblot analysis using an antibody specific for phosphorylated Thr-286. We have previously demonstrated that recognition of cyclin D1 by this antibody is strictly dependent upon phosphorylation of Thr-286 (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar). FLAG-D1, FLAG-D1-T286A, or FLAG-D1-V290/295A was precipitated from cells with the M2 monoclonal antibody. Although the D1-T286A mutant was not phosphorylated, both wild type cyclin D1 and D1-V290/295A were phosphorylated on Thr-286 (Fig. 2A). In fact D1-V290/295A phosphorylation was increased relative to wild type cyclin D1, suggesting decreased turnover of this mutant cyclin D1 protein (see Fig. 3). These results are consistent with the notion that hydrophobic residues between amino acids 290 and 295 direct cyclin D1-CRM1 association. Furthermore, these results demonstrate that phosphorylation of cyclin D1 at Thr-286 alone is insufficient to direct CRM1-mediated cyclin D1 nuclear export in the absence of these hydrophobic residues.Fig. 3Increased stability of constitutively nuclear cyclin D1 mutants. A, NIH-3T3 cells stably overexpressing the indicated cyclin D1 proteins were treated with cycloheximide (CHX) for the indicated intervals. Lysates prepared from the respective cells were subjected to Western analysis using the cyclin D1 monoclonal antibody. Results shown are representative of multiple independent experiments. B, wild type cyclin D1 or D1-V290/295A was precipitated from lysates prepared from cells treated with cycloheximide as indicated, and decay of phosphorylated cyclin D1 proteins was assessed by immunoblot with the phospho-T286 antibody. C, HA-ubiquitin co-transfected with either cyclin D1 or D1-V290/295A was immunoprecipitated from cells in the presence or absence of MG132, and ubiquitinated cyclin D1 was detected by Western blot. poly-Ub, poly-ubiquitin laddering.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next determined whether these alanine substitutions specifically interfere with CRM1 binding or whether they have a general effect on cyclin D1 folding that would be reflected in its capacity to associate with other known cyclin D1 interacting proteins or, alternatively, in its capacity to support CDK4 catalytic activity. We first assessed the association of D1-V290/295A with GSK-3β. We have previously demonstrated that kinase-defective GSK-3β will form stable complexes with cyclin D1 in insect Sf9 cells (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). Insect Sf9 cells were infected with baculoviruses encoding kinase-defective GSK-3β along with cyclin D1, D1-T286A, or cyclin D1-V290/295A. GSK-3β-cyclin D1 complexes were isolated from lysates prepared from these cells by precipitation with a GSK-3β-specific monoclonal antibody. Immunoblot analysis confirmed the presence of D1-V290/295A within GSK-3β precipitates (Fig. 2B). Overexpression of GSK-3β will drive wild type cyclin D1 into the cytoplasm (15Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar, 17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar), whereas the phosphorylation-deficient D1-T286A mutant is refractory to this enforced nuclear export. Because D1-V290/295A retained both binding to and phosphorylation by GSK-3β, it was important to determine whether D1-V290/295A was refractory to GSK-3β-mediated nuclear export. We examined the subcellular localization of cyclin D1 and D1-V290/295A in asynchronously proliferating cells transfected with GSK-3β. As shown in Fig. 2C, cyclin D1 was efficiently shuttled out of the nucleus and cytoplasmically localized in the presence of GSK-3β. In contrast, D1-V290/295A remained nuclear and is thus refractory to GSK-3β-mediated nuclear export (Fig. 2C, panels c and d). GSK-3β expression was confirmed by indirect immunofluorescence in parallel (data not shown). We subsequently assessed the capacity of D1-V290/295A to associate with and activate CDK4 as well as to retain binding to p21Cip1. Similar to wild type cyclin D1 and D1-T286A, cyclin D1-V290/295A assembled with CDK4 and supported CDK4-dependent phosphorylation of Rb (data not shown). Moreover, D1-V290/295A also associated with p21Cip1 (Fig. 2D), indicating that the alanine substitutions have not significantly perturbed the structural or functional integrity of cyclin D1-V290/295A. These data support the hypothesis that residues 290-295 of cyclin D1 mediate CRM1 association and together with phosphorylated Thr-286 constitute the cyclin D1 nuclear export signal. Increased Stability of Constitutively Nuclear Cyclin D1 Mutants—Our previous work has suggested that cyclin D1 proteolysis is a cytoplasmic event. This conclusion was based on the increased stability of constitutively nuclear cyclin D1-T286A. However, it was not possible to determine the role of Thr-286 phosphorylation versus nuclear export in mediating cyclin D1 proteolysis. In characterizing D1-V290/295A, we noted that it accumulated as a Thr-286-phosphorylated protein (Fig. 2A, compare lanes 1 and 3), suggesting that phosphorylation is not sufficient to trigger proteolysis of this nuclear protein. Using cycloheximide to inhibit nascent protein synthesis (30Rimerman R.A. Gellert-Randleman A. Diehl J.A. J. Biol. Chem. 2000; 275: 14736-14742Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we investigated the rate of D1-V290/295A degradation versus that of either wild type cyclin D1 or the D1-T286A mutant (Fig. 3A). Our results revealed that both D1-V290/295A and D1-T286A have increased protein stability in comparison with wild type cyclin D1. To confirm reduced Thr-286 proteolysis of phosphorylated D1-V290/295A, we measured decay of phosphorylated isoforms of wild type cyclin D1 and D1-V290/295A. Although the half-life of Thr-286 phosphorylated cyclin D1 was less than 15 min, the half-life of phospho-D1-V290/295A was greatly extended (Fig. 3B). Moreover, to corroborate the increased stability of D1-V290/295A relative to wild type cyclin D1, we assessed ubiquitination of cyclin D1 versus D1-V290/295A. Either wild type or D1-V290/295A was co-transfected with HA-ubiquitin into NIH-3T3 cells. Transfected cells were treated with either vehicle or MG132 to inhibit the 26 S proteasome. Cells were then harvested, and lysates were subjected to immunoblotting with the cyclin D1 monoclonal antibody (Fig. 3C). Wild type cyclin D1 alone exhibited the characteristic poly-ubiquitin laddering (poly-Ub) recognized for ubiquitinated proteins in the presence of MG132 (Fig. 3C, lane 2). No higher molecular weight isoforms of D1-V290/295A were detected either in the presence or absence of MG132. These data demonstrate that disruption of CRM1 association promotes the accumulation of Thr-286 phosphorylated cyclin D1 that is inaccessible to and consequently not degraded via cytoplasmic 26 S proteasomes. Loss of CRM1 Binding Increases the Transforming Potential of Cyclin D1—Cells engineered to overexpress cyclin D1 display a contracted G1 interval (18Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.Y. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar). These cells, however, do not form foci, grow in soft agar, or promote tumor formation in immunocompromised mice (18Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.Y. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar). As shown previously in our laboratory, a cyclin D1 mutant that cannot be phosphorylated at Thr-286 and is thus constitutively nuclear promotes cellular transformation (17Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (458) Google Scholar). This result suggests that disruption of cyclin D1 nuclear export is an oncogenic event. We therefore considered the possibility that the D1-V290/295A mutant, which unlike D1-T286A retains phosphorylation at Thr-286, might exhibit an increased potential to drive cell transformation relative to wild type cyclin D1. For these experiments NIH-3T3 cells were co-transfected with vectors encoding either wild type FLAG-tagged cyclin D1 or the specified cyclin D1 mutant and a vector encoding puromycin as a selectable marker. Transfectants selected in puromycin were then pooled to eliminate potential clonal variation when assessing transformation. NIH-3T3 cells (data not shown) or NIH-3T3 derivatives engineered to overexpress either wild type FLAG-tagged or mutant cyclin D1 isoforms were assayed for characteristics of cell transformation: foci formation (refractory to contact inhibition) and growth in soft agar (capable of anchorage-independent growth). Over the course of five independent experiments, all NIH-3T3 cells overexpressing the C-terminal cyclin D1 mutants D1-T286A and D1-V290/295A reproducibly formed numerous foci (Fig. 4A; data not shown). Consistent with these results, unlike wild type NIH-3T3 (data not shown) and D1-3T3 cells, which were incapable of significant growth in soft agar, NIH-3T3 cell lines overexpressing constitutively nuclear C-terminal cyclin D1 mutants D1-T286A (used as positive control) and D1-V290/295A reproducibly grew in soft agar, forming numerous and robust colonies (Fig. 4, B and C (quantitation)). Our data support the model wherein mutations in the CRM1 binding site and/or loss of Thr-286 phosphorylation in wild type cyclin D1 leads to a constitutively nuclear cyclin D1 with increased capacity to drive neoplastic transformation and, as a result, a loss in G1/S homeostasis (Fig. 5).Fig. 5Cell transformation results from inhibition of cyclin D1 nuclear export. Wild type cyclin D1 is phosphorylated at Thr-286 (circled “P”) in late G1 phase, allowing for CRM1 recognition and binding that triggers the rapid nuclear export of cyclin D1-CDK4 complexes in S phase (top). Mutation of phosphorylatable Thr-286 or mutations directly within the CRM1 binding site of cyclin D1 results in abrogated CRM1 binding and inhibition of cyclin D1 nuclear export. Expression of constitutively nuclear C-terminal cyclin D1 mutants results in cellular transformation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cyclin D1 localization is driven by the competing processes of nuclear import and export. Although the mechanisms of cyclin D1 nuclear import remain poorly characterized, it is now clear that CRM1-dependent nuclear export drives cyclin D1 complexes into the cytoplasm during S phase. CRM1 directs nuclear export of target proteins via direct binding to leucine/hydrophobic stretches of amino acids. However, the exact spacing of the leucine/hydrophobic patch is variable making it difficult to identify CRM1 binding sites by scanning the primary sequence of a given putative substrate. Although two stretches of residues closely conformed to previously documented nuclear export signal motifs, residues 87-94 (RFLSLEPL) and residues 290-295, the 290-295 sequence was an attractive target given its proximity to the site of GSK-3β-mediated phosphorylation, Thr-286, and its highly conserved nature among all three D-type cyclins. Mutation of valines and isoleucines to alanines in this region abrogates CRM1 binding and promotes nuclear retention without perturbing phosphorylation of Thr-286. This mutant, D1-V290/295A, accumulates in the nucleus as a Thr-286-phosphorylated protein. In contrast, the phosphorylated form of wild type cyclin D1 is highly labile and is detectable in the cytoplasm (9Alt J.R. Gladden A.B. Diehl J.A. J. Biol. Chem. 2002; 277: 8517-8523Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Taken together, these data identify residues 290-295 as the CRM1 binding site in cyclin D1. Our data demonstrate that a hydrophobic patch within the C terminus of cyclin D1 mediates CRM1 binding. Mutations within this region do not perturb GSK-3β-mediated phosphorylation of cyclin D1 at Thr-286, a modification that increases the propensity for CRM1 recognition and binding. As our results indicate, however, phosphorylation in itself is insufficient for cyclin D1 nuclear export or degradation, which is consistent with cytoplasmic proteolysis of cyclin D1. Thus, in the absence of an intact CRM1 binding site, CRM1 fails to associate with cyclin D1, and the resulting protein is more stabile and constitutively nuclear. These data suggest that the C terminus of cyclin D1 is a critical modulator of cyclin D1 nuclear export and might therefore represent a potential “hot spot” for the activation of mutations in cyclin D1 that ultimately contribute to cancer genesis. In support of this notion, we have recently identified a splice variant of cyclin D1, referred to as cyclin D1b, which lacks the C-terminal residues that direct GSK-3β phosphorylation and CRM1-dependent nuclear export (20Lu F. Gladden A.B. Diehl J.A. Cancer Res. 2003; 63: 7056-7061PubMed Google Scholar). As with D1-V290/295A described herein, cyclin D1b is constitutively nuclear and can drive cell transformation (20Lu F. Gladden A.B. Diehl J.A. Cancer Res. 2003; 63: 7056-7061PubMed Google Scholar, 31Solomon D.A. Wang Y. Fox S.R. Lambeck T.C. Giesting S. Lan Z. Senderowicz A.M. Knudsen E.S. J. Biol. Chem. 2003; 278: 30339-30347Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Furthermore, we have found that this protein is expressed in a significant fraction of primary cancers, highlighting the significance of maintaining temporal control of cyclin D1 subcellular localization for normal cell growth and proliferation (20Lu F. Gladden A.B. Diehl J.A. Cancer Res. 2003; 63: 7056-7061PubMed Google Scholar). We thank C. J. Sherr for providing NIH-3T3 cells and J. Woodgett for GSK-3β cDNAs." @default.
• W1982074920 created "2016-06-24" @default.
• W1982074920 creator A5028194905 @default.
• W1982074920 creator A5035025572 @default.
• W1982074920 date "2004-12-01" @default.
• W1982074920 modified "2023-10-03" @default.
• W1982074920 title "C-terminal Sequences Direct Cyclin D1-CRM1 Binding" @default.
• W1982074920 cites W1963705823 @default.
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