Title: Construction of a Cyclin D1-Cdk2 Fusion Protein to Model the Biological Functions of Cyclin D1-Cdk2 Complexes
Abstract: Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr160. Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr160 and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr160. These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGFβ-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes. Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr160. Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr160 and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr160. These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGFβ-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes. Cyclin D1 provides a critical link between signaling initiated by extracellular growth factors and cell cycle regulation. Cyclin D1 levels in the mammary gland are regulated by steroid hormones and polypeptide growth factors, suggesting a unique role for cyclin D1 in mammary development (1Sicinski P. Donaher J.L. Parker S.B. Li T. Fazeli A. Gardner H. Haslam S.Z. Bronson R.T. Elledge S.J. Weinberg R.A. Cell. 1995; 82: 621-630Abstract Full Text PDF PubMed Scopus (889) Google Scholar). Consistent with an important role for cyclin D1 in mammary gland function, mice lacking cyclin D1 fail to undergo normal lobuloalveolar development during pregnancy (1Sicinski P. Donaher J.L. Parker S.B. Li T. Fazeli A. Gardner H. Haslam S.Z. Bronson R.T. Elledge S.J. Weinberg R.A. Cell. 1995; 82: 621-630Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 2Fantl V. Stamp G. Andrews A. Rosewell I. Dickson C. Genes Dev. 1995; 9: 2364-2372Crossref PubMed Scopus (602) Google Scholar). Cyclin D1 regulates mammary development through the formation of catalytically active complexes with the cyclin-dependent kinase Cdk4 1The abbreviations used are: Cdk, cyclin-dependent kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; MOPS, 4-morpholinepropanesulfonic acid; Rb, retinoblastoma tumor suppressor protein; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PCNA, proliferating cell nuclear antigen; TGF, tumor growth factor.1The abbreviations used are: Cdk, cyclin-dependent kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; MOPS, 4-morpholinepropanesulfonic acid; Rb, retinoblastoma tumor suppressor protein; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PCNA, proliferating cell nuclear antigen; TGF, tumor growth factor. (3Muraoka 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 (104) Google Scholar). Mice lacking the assembly factor p27 fail to form active cyclin D1-Cdk4 complexes and exhibit a phenotype similar to that observed in cyclin D1-deficient mice, including impaired lobuloalveolar differentiation and the inability to lactate (4Muraoka 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).Overexpression of cyclin D1 in the mouse mammary gland under the control of the MMTV promoter induces mammary hyperplasia and tumor formation, indicating that cyclin D1 levels control the rate of mammary epithelial cell proliferation (5Wang T.C. Cardiff R.D. Zukerberg L. Lees E. Arnold A. Schmidt E.V. Nature. 1994; 369: 669-671Crossref PubMed Scopus (890) Google Scholar). Cyclin D1 overexpression likely also contributes to human mammary tumorigenesis since cyclin D1 is overexpressed in ∼50% of human breast cancers (6Sutherland R.L. Musgrove E.A. Breast Cancer Res. 2002; 4: 14-17Crossref PubMed Scopus (102) Google Scholar, 7Buckley M.F. Sweeney K.J. Hamilton J.A. Sini R.L. Manning D.L. Nicholson R.I. deFazio A. Watts C.K. Musgrove E.A. Sutherland R.L. Oncogene. 1993; 8: 2127-2133PubMed Google Scholar). Given the role that cyclin D1 plays in mammary tumorigenesis, it is critical to understand the mechanisms by which cyclin D1 promotes cell proliferation and transformation.Active cyclin D1-Cdk4 complexes phosphorylate the retinoblastoma tumor suppressor protein (Rb) releasing the repression of E2F-dependent transcription and allowing the expression of genes required for progression into the S-phase of the cell cycle. A mutant form of Cdk4 that is not inhibited by p16 induces tumorigenesis, indicating that deregulated cyclin D-Cdk4 complexes are capable of driving cancer formation (8Rane S.G. Cosenza S.C. Mettus R.V. Reddy E.P. Mol. Cell. Biol. 2002; 22: 644-656Crossref PubMed Scopus (131) Google Scholar). Cyclin D1 also indirectly promotes cell proliferation by sequestering p21 and p27, resulting in the activation of Cdk2 (9Perez-Roger I. Kim S.H. Griffiths B. Sewing A. Land H. EMBO J. 1999; 18: 5310-5320Crossref PubMed Scopus (280) Google Scholar, 10Bouchard C. Thieke K. Maier A. Saffrich R. Hanley-Hyde J. Ansorge W. Reed S. Sicinski P. Bartek J. Eilers M. EMBO J. 1999; 18: 5321-5333Crossref PubMed Scopus (405) Google Scholar). It is generally assumed that cyclin D1 drives cell proliferation and oncogenic transformation through these two mechanisms. Interestingly, cyclin D1-Cdk2 complexes were observed in earlier studies (11Dou Q.P. Molnar G. Pardee A.B. Biochem. Biophys. Res. Commun. 1994; 205: 1859-1868Crossref PubMed Scopus (12) Google Scholar, 12Dulic V. Drullinger L.F. Lees E. Reed S.I. Stein G.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11034-11038Crossref PubMed Scopus (284) Google Scholar). In one study, cyclin D1-Cdk2 complexes were detected in 9 of 13 human mammary carcinomas (13Sweeney K.J. Swarbrick A. Sutherland R.L. Musgrove E.A. Oncogene. 1998; 16: 2865-2878Crossref PubMed Scopus (70) Google Scholar). However, cyclin D1-Cdk2 complexes have been assumed to be catalytically inactive based on a report showing that cyclin D1-Cdk2 complexes expressed in Sf9 insect cells using the baculovirus system were not phosphorylated by cyclin-dependent kinase-activating kinase (CAK) containing cyclin H and Cdk7 (14Higashi H. Suzuki-Takahashi I. Saitoh S. Segawa K. Taya Y. Okuyama A. Nishimura S. Kitagawa M. Eur. J. Biochem. 1996; 237: 460-467Crossref PubMed Scopus (32) Google Scholar).In the present study we show that cyclin D1-Cdk2 complexes exist in both human mammary carcinoma cell lines and mouse mammary epithelial cells. In fact, these cyclin D1-Cdk2 complexes are phosphorylated on the activating CAK phosphorylation site of Cdk2, Thr160. Isolated cyclin D1-Cdk2 complexes phosphorylate histone H1 and Rb on sites preferred by Cdk2, as well as on sites preferred by Cdk4. Furthermore, we have constructed a cyclin D1-Cdk2 fusion protein to model the biological functions of cyclin D1-Cdk2 complexes. The cyclin D1-Cdk2 fusion protein phosphorylates Rb in vitro on sites preferred by Cdk2 and Cdk4. Stable expression of the cyclin D1-Cdk2 fusion protein confers anchorage-independent cell proliferation and resistance to the growth inhibitory effects of TGFβ. These data suggest that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 overexpression in cancers and may be a useful target for anti-cancer therapeutics.MATERIALS AND METHODSCell Culture, [3H]Thymidine Incorporation Assays, and Soft Agar Assays—NMuMG, MDA-MB-231, and HaCaT cells were propagated as described previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar). HEK 293, HCT116, and MDA-MB-361 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium (Invitrogen, Life Technologies, Inc.) supplemented with 10% fetal bovine serum. p21-null HCT116 cells were obtained from Dr. Bert Vogelstein, Johns Hopkins, Baltimore, MD, and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. [3H]Thymidine incorporation assays and soft agar assays were performed as described previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar). Images of soft agar colonies were captured using an Olympus CK40 inverted microscope and an Olympus DP10 digital camera.Construction of Recombinant Adenoviruses, Recombinant Retroviruses, Stable Cell Lines, and Transient Transfections—Cdk2-His6, His6-p27, FLAG-Cyclin D1, and Cdk4-His6 were constructed by polymerase chain reaction using the primers -Cdk2-His6: 5′-TTTTGGATCCATGGAGAACTTCCAAAAG-3′; 5′-TTTTGAATTCTCAATGATGATGATGATGATGGAGTCGTTGATGGGGTAC-3′, His6-p27: 5′-TTTTGGATCCATGCATCATCATCATCATCATTCAAACGTGCGAGTGTCTAAC-3′; 5′-TTTTGAATTCTTACGTTTGACGTCTTCTGAG-3′, FLAG-Cyclin D1: 5′-TTTTGAATTCCCACCATGGACTATAAGGACGATGATGACAAAGAACACCAGCTCCTGTGCTGC-3′; 5′-TTTTCTCGAGTCAGATGTCCACATCTCG-3′, and Cdk4-His6: 5′-TTTTGGATCCATGGCTACCTCTCGATATGAG-3′; 5′-TTTTGAATTCTCAATGATGATGATGATGATGCTCCGGATTACCTTC ATC-3′, respectively, cloned into pcDNA3 (Invitrogen), and the inserts were verified by DNA sequencing. These inserts were subcloned into the pAd-Track-CMV vector and adenoviruses were prepared using the pAd-Easy system (16He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3229) Google Scholar). Adenoviruses produced using the pAdTrack-CMV vector contain two reading frames, each driven by a separate CMV promoter. The first reading frame encodes green fluorescent protein (GFP) and the second reading frame encodes the gene of interest. Adenoviruses were propagated in HEK 293 cells and viral titers were determined using a variation on the plaque assay in which the number of infectious particles was followed by the number of foci of GFP fluorescence produced in HEK 293 cells. Adenoviral infection of MDA-MB-231, MDA-MB-361, or NMuMG target cells was performed in serum-free medium in the presence of 5 μg/ml polybrene (17Doebis C. Ritter T. Brandt C. Schonberger B. Volk H.D. Seifert M. Transpl. Immunol. 2002; 9: 323-329Crossref PubMed Scopus (22) Google Scholar) at a multiplicity of infection (MOI) of 100. At this MOI ∼50% of MDA-MB-231, 25% of MDA-MB-361, and 25% of the NMuMG cells were strongly positive for GFP fluorescence.Recombinant retroviruses and stable cell lines were prepared as described previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar) using the pBabe retroviral vector encoding the puromycin resistance gene (18Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1892) Google Scholar). To construct the cyclin D1-Cdk2 fusion gene, cyclin D1 with the stabilizing T286A mutation (3Muraoka 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 (104) Google Scholar) was amplified using the primers 5′-TTTTGAATTCCCACCATGGACTATAAGGACGATGATGACAAAGAACACCAGCTCCTGTGCTGC-3′; 5′-ACCTCCACCACCGGATCCTCCACCTCCAGAACCTCCACCACCGATGTCCACATCTCGCACGTC-3′ and cloned into the EcoRI and BamHI sites of pSP72 (Promega). Cdk2 was amplified using the primers 5′-GGTGGTGGAGGTTCTGGAGGTGGAGGATCCGGTGGTGGAGGTGAGAACTTCCAAAAGGTGGAAAAG-3′; 5′-TTTTCTCGAGTCAGTGGTGGTGGTGGTGGTGGAGTCGAAGATGGGGTACTGG-3′, and cloned into the BamHI and XhoI sites to form the cyclin D1-Cdk2 fusion gene between the EcoRI and XhoI sites. The EcoRI-XhoI fragment was cloned into the EcoRI and XhoI sites of pcDNA3, verified by DNA sequencing, and the insert cloned into pBabe-Puro to construct recombinant retroviruses encoding the cyclin D1-Cdk2 fusion gene. Point mutants of the cyclin D1-Cdk2 fusion gene were prepared in the pcDNA3 vector using QuikChange PCR (Stratagene) and verified by DNA sequencing. The K114E mutation of cyclin D1 (19Hinds P.W. Dowdy S.F. Eaton E.N. Arnold A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 709-713Crossref PubMed Scopus (453) Google Scholar) and the D145N mutation of Cdk2 (20van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (970) Google Scholar) were introduced by PCR using the primer sets K114E: 5′-GGCCTCTAAGATGGAGGAGACCATTCCCTTG-3′; 5′-CAAGGGAATGGTCTCCTCCATCTTAGAGGCC-3′, and D145N: 5′-CAAGCTAGCAAACTTTGGACTAGCCAGAG-3′; 5′-CTCTGGCTAGTCCAAAGTTTGCTAGCTTG-3′, respectively.Transient transfections in HEK 293 cells were carried out as described previously (21Law B.K. Waltner-Law M.E. Entingh A.J. Chytil A. Aakre M.E. Norgaard P. Moses H.L. J. Biol. Chem. 2000; 275: 38261-38267Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) using LipofectAMINE (Invitrogen, Life Technologies, Inc.). Transient transfections in HaCaT cells were performed using FuGENE (Roche Applied Science) according to the manufacturer's instructions. E2F transcriptional reporter assays were performed as described previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar).Isolation of Cdk2 and Cdk4 Complexes by Immobilized Metal Affinity Chromatography, Immunoprecipitation, and Protein Kinase Assays— Forty-eight hours post-infection with recombinant adenoviruses, cells were lysed in TALON buffer (10 mm MOPS, pH 7.0, 10% glycerol, 100 mm KCl, 10 nm microcystin, 3 mm imidazole, 5 mm MgCl2) containing 0.2% Triton X-100, scraped from the plates with a cell scraper, and sonicated. The lysates were clarified by centrifugation and the supernatants were mixed with TALON resin (Clontech) for 3 h at 4 °C. The resin was washed three times with TALON buffer containing 50 mm imidazole and proteins bound to the resin were eluted with 400 mm imidazole.In sequential TALON-anti-FLAG-agarose purifications, the imidazole-eluted proteins were diluted 10-fold and incubated with anti-FLAG-agarose (Sigma) for 3 h at 4 °C. The beads were washed four times and eluted with 200 μm FLAG peptide (Sigma) for 45 min at 25 °C.Protein assays, immunoprecipitation, kinase assays, and immunoblot analyses were performed as described previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar). Kinase assay results were visualized using a phosphorimager and ImageGuage software (Fuji). The sources of many of the antibodies used here were listed previously (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar, 21Law B.K. Waltner-Law M.E. Entingh A.J. Chytil A. Aakre M.E. Norgaard P. Moses H.L. J. Biol. Chem. 2000; 275: 38261-38267Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Law B.K. Norgaard P. Gnudi L. Kahn B.B. Poulson H.S. Moses H.L. J. Biol. Chem. 1999; 274: 4743-4748Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Law B.K. Norgaard P. Moses H.L. J. Biol. Chem. 2000; 275: 10796-10801Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Anti-phospho-Rb antibodies specific for the Phospho-Ser780, -Ser807/Ser811, -Ser795 sites, and an antibody recognizing phospho-Tyr15 of Cdk2 were purchased from Cell Signaling Technologies. Monoclonal antibodies recognizing the FLAG (M2) and hexahistidine (H5) tags were obtained from Sigma and Qiagen, respectively. Antibodies to cyclin E (sc-481), PCNA (sc-56), and actin (sc-8432), and the cyclin D1 antibody (sc-450) used for immunoprecipitation and kinase assays, were from Santa Cruz Biotechnology, Inc.Identification of Cdk2 Complex Components by Mass Spectrometry— Proteins eluted from anti-FLAG-agarose resin (above) were separated by SDS-PAGE and the gel was stained with colloidal Coomassie (Invitrogen). Protein bands corresponding to Cdk2 complex components were identified by the Vanderbilt Proteomics Laboratory, Mass Spectrometry Research Center. Briefly, bands were excised and subjected to in-gel trypsin digestion. The masses of the eluted peptides were determined by matrix-assisted laser desorption time of flight (MALDI-TOF) and TOF/TOF tandem mass spectrometry using an Applied Biosystems Voyager 4700 mass spectrometer. Proteins corresponding to the bands of interest were identified by interrogating the Swiss-Prot and NCBInr protein databases with the acquired peptide mass maps and fragmentation spectra using GPS Explorer software (Applied Biosystems) running the MASCOT search engine (Matrix Science). Searches were performed with mass accuracies of less than 20 ppm, allowing for 1 missed cleavage, complete carbamidomethylation of cysteine sulfhydryls, and partial oxidation of methionine residues.RESULTSCharacterization of Cyclin D1-Cdk2 Complexes from Human Carcinoma and Mouse Mammary Epithelial Cells—We previously showed that treatment of mouse mammary epithelial cells and human mammary carcinoma cells with TGFβ + rapamycin alters the subunit composition of Cdk2 complexes and alters Cdk2 intracellular localization (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar, 24Brown K.A. Roberts R.L. Arteaga C.L. Law B.K. Breast Cancer Res. 2004; 6: R130-R139Crossref PubMed Google Scholar). To systematically study TGFβ and rapamycin regulation of Cdk2 complexes we constructed a recombinant adenovirus encoding hexahistidine-tagged Cdk2 (Cdk2-His6) to allow the rapid purification and analysis of intact Cdk2 complexes. An adenovirus encoding GFP was used as a control for the purifications. Cdk2 complexes isolated from human mammary MDA-MB-231 carcinoma cells consistently contained cyclin D1 (Fig. 1A). The levels of cyclin D1-Cdk2 complexes were not altered by TGFβ + rapamycin treatment. Similar results were obtained with human mammary MDA-MB-361 carcinoma cells (data not shown). A previous study demonstrated the presence of cyclin D1-Cdk2 complexes in human mammary carcinoma cell lines (13Sweeney K.J. Swarbrick A. Sutherland R.L. Musgrove E.A. Oncogene. 1998; 16: 2865-2878Crossref PubMed Scopus (70) Google Scholar). However, whether cyclin D1-Cdk2 complexes play a role in cell cycle regulation in mammary carcinoma cells or mammary epithelial cells has not been explored.To determine whether endogenous cyclin D1-Cdk2 complexes are present in mouse mammary epithelial cells, coimmunoprecipitation experiments were performed in mouse mammary NMuMG cells (Fig. 1B). These experiments demonstrated that cyclin D1-Cdk2 complexes were present in NMuMG cells and that their levels were unaffected by TGFβ, rapamycin, or TGFβ + rapamycin treatment. Strikingly, Cdk2 associated with cyclin D1 was phosphorylated on the cyclin-dependent kinase activating kinase site, Thr160, suggesting that cyclin D1-Cdk2 complexes may be catalytically active.In addition to acting as a kinase, cyclin D1-Cdk2 complexes might also regulate the cell cycle indirectly by sequestering p21 and p27. There is extensive evidence that cyclin D1-Cdk4 complexes regulate the cell cycle through this mechanism; however, cyclin D1-Cdk2 complexes have not been shown to perform a similar function. To determine the subunit structure of cyclin D1-Cdk2 complexes, NMuMG cells were co-infected with recombinant adenoviruses encoding FLAG-epitope-tagged cyclin D1 and Cdk2-His6. Cyclin D1-Cdk2 complexes were purified by sequential chromatography using TALON resin to bind His6-tagged Cdk2 and anti-FLAG-agarose resin to bind cyclin D1-containing complexes. Purified cyclin D1-Cdk2 complexes from cells treated with TGFβ + rapamycin (lane 3) or left untreated (lane 2), or a control purification using GFP adenovirus (lane 1), were resolved by SDS-PAGE and visualized by colloidal Coomassie staining (Fig. 1C). Individual bands were excised, subjected to trypsin digestion, and the peptides analyzed by MALDI-TOF mass spectrometry. These analyses indicated that the cyclin D1 complexes contained proliferating cell nuclear antigen (PCNA) and p21 in a near stoichiometric ratio as judged by Coomassie staining. Interestingly, TGFβ + rapamycin treatment dramatically increased the amount of p27 present in cyclin D1-Cdk2 complexes. This result is consistent with our previous study (15Law B.K. Chytil A. Dumont N. Hamilton E.G. Waltner-Law M.E. Aakre M.E. Covington C. Moses H.L. Mol. Cell. Biol. 2002; 22: 8184-8198Crossref PubMed Scopus (90) Google Scholar), but extends these results by showing that TGFβ + rapamycin-induced p27 binding is independent of the cyclin (E, A, or D1) associated with Cdk2. The observation that cyclin D1-Cdk2 complexes bind both p21 and p27 suggests that, like cyclin D1-Cdk4 complexes, cyclin D1-Cdk2 complexes sequester p21 and p27 and may indirectly activate cyclin E-Cdk2 and cyclin A-Cdk2 complexes.p21 Stabilizes, but Is Not Required for the Formation of Cyclin D1-Cdk2 Complexes—The experiment in Fig. 1C involved overexpression of both cyclin D1 and Cdk2, so we examined NMuMG cells and cancer cell lines for the presence of endogenous cyclin D1-Cdk2 complexes. Co-immunoprecipitation experiments demonstrated that cyclin D1-Cdk2 complexes are present at relatively high levels in human HCT116 colorectal carcinoma cells and NMuMG cells, and at lower levels in T98G glioblastoma cells or SW480 colorectal carcinoma cells (Fig. 2A). In addition to cyclin D1, Cdk2 complexes from NMuMG and HCT116 cells contained high levels of p21 and PCNA. In contrast, Cdk2 complexes from T98G and SW480 cells contained low levels of cyclin D1, p21, and PCNA. These results are consistent with the results in Fig. 1C, and suggest that cyclin D1-Cdk2 complexes in rapidly growing NMuMG and HCT116 cells are part of a quaternary complex containing cyclin D1, Cdk2, p21, and PCNA. Interestingly, Cdk2 immunoprecipitated from all four cell lines associated with similar amounts of E2F4, p107, and p130. This observation suggests that different subsets of Cdk2-containing complexes may be differentially regulated by cellular levels of p21, p27, or PCNA.Fig. 2p21 stabilizes, but is not required for, the formation of cyclin D1-Cdk2 complexes.A, extracts from the indicated cell lines were immunoprecipitated (IP) with antibodies specific for Cdk2, E2F4, or normal rabbit IgG (Rab. IgG) as a control. The immunoprecipitates were analyzed by immunoblot (IB) with the indicated antibodies. B, cell extracts were prepared from rapidly growing HCT116 cells (+ Serum) or HCT116 cells deprived of serum for 14 h (– Serum) that were either wild type for p21 (p21+/+) or p21-null (p21–/–). Equal amounts of protein were immunoprecipitated (IP) with antibodies to Cdk2, or normal rabbit serum (Rab. IgG) as a control, and the crude extracts and Cdk2 immunoprecipitates corresponding to each sample were analyzed by immunoblot with the indicated antibodies.View Large Image Figure ViewerDownload (PPT)p21 and p27 act as assembly factors for the formation of cyclin D1-Cdk4 complexes (25LaBaer 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 (1209) Google Scholar, 26Cheng 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 (965) Google Scholar). However, it is unclear whether p21 or p27 is required for the formation of cyclin D1-Cdk2 complexes. To address this question, extracts from parental or p21-null HCT116 cells were examined for the presence of cyclin D1-Cdk2 complexes in coimmunoprecipitation experiments (Fig. 2B). Cyclin D1-Cdk2 complexes were present in both parental and p21-null HCT116 cells that were either rapidly growing or rendered quiescent by serum deprivation. The levels of cyclin D1-Cdk2 complexes were slightly decreased in p21-null HCT116 cells suggesting that although p21 is not absolutely required for the formation of cyclin D1-Cdk2 complexes, p21 may stabilize these complexes. Cdk2 complexes from neither parental nor p21-null HCT116 cells contained significant amounts of p27, indicating that p27 did not substitute as a cyclin D1-Cdk2 assembly factor in the absence of p21 (data not shown). p21 deletion prevented PCNA binding to cyclin D1-Cdk2 complexes. This was expected based on previous studies showing that p21 binds Cdk2 through its N terminus while p21 binds PCNA through its C terminus and serves to bridge Cdk2 and PCNA (27Nakanishi M. Robetorye R.S. Adami G.R. Pereira-Smith O.M. Smith J.R. EMBO J. 1995; 14: 555-563Crossref PubMed Scopus (172) Google Scholar, 28Nakanishi M. Robetorye R.S. Pereira-Smith O.M. Smith J.R. J. Biol. Chem. 1995; 270: 17060-17063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). p21 deletion did not alter the levels of Cdk2/p107/E2F4 complexes, supporting the notion that basal levels of p21 in HCT116 cells regulate the subunit composition of cyclin D1-Cdk2 complexes but not E2F/Cdk2 complexes.Cyclin D1 and Cdk2 Cooperate to Induce Rb Phosphorylation and Activate E2F-dependent Transcription—Cyclin D1-Cdk2 complexes might regulate cell cycle progression indirectly by sequestering p21 and p27 as discussed above or might regulate cell proliferation directly by phosphorylating important cell cycle regulators such as the pocket proteins Rb, p130, and p107. To assay the kinase activity of cyclin D1-Cdk2 complexes, NMuMG cells were infected with adenovirus encoding Cdk2-His6, and cyclin D1-Cdk2 complexes were isolated by chromatography on TALON resin followed by immunoprecipitation with anti-cyclin D1 antibodies. These complexes exhibited kinase activity when assayed using Histone H1 as the substrate and exhibited Cdk2 phosphorylation on Thr160 (Fig. 3A).Fig. 3Cyclin D1 and Cdk2 form catalytically active kinase complexes that phosphorylate Rb and activate E2F-dependent transcription.A, NMuMG cells were infected with adenoviruses (Ad) encoding GFP or Cdk2-His6 as indicated and incubated 48 h. Cell extracts were prepared and purified over TALON resin. Proteins eluted from the TALON resin were immunoprecipitated (IP) with antibodies to cyclin D1 or a nonspecific isotype control antibody (NS IgG1). Immunoprecipitates were assayed for kinase activity using histone H1 as the substrate. Kinase assay results were visualized using a phosphorimager. The kinase reactions were also analyzed by immunoblot with antibodies specific for cyclin D1, Cdk2, or Cdk2 phosphorylated on Thr160 (P-Cdk2(T160)). B, HaCaT cells were transfected with empty vector (pcDNA3), or pcDNA3 encoding Cdk2-His6, cyclin D1 (D1), cyclin D1(T286A) (D1(T286A)), or FLAG-tagged cyclin D1(T286A) (Flag-D1(T286A)) in the indicated combinations. Cell extracts were prepared and the proteins analyzed by immunoblotting (top panel) with antibodies specific for Rb, Cdk2, cyclin D1, actin, or Rb-phosphorylated on Ser249/Thr252 (P-Rb(S249/T252). The same cell extracts were immunoprecipitated with antibody to the
Publication Year: 2004
Publication Date: 2004-11-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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