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• W2081736214 abstract "Mitogenic stimulation leads to activation of G1 cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G0/G1 and G1/S transitions of the cell cycle. However, the individual role of G1 cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 “form 3,” which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle. Mitogenic stimulation leads to activation of G1 cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G0/G1 and G1/S transitions of the cell cycle. However, the individual role of G1 cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 “form 3,” which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle. cyclin-dependent kinase fetal bovine serum 3-(cyclohexylamino)propanesulfonic acid poly(ADP-ribose) polymerase carboxymethyl fluorescein diacetate succinyl ester G1 cyclin-dependent kinases (CDKs)1 regulate progression through the G0/G1 transition and entry into the S-phase of the cell cycle following activation by mitogenic signaling pathways (1Peeper D.S. Upton T.M. Ladha M.H. Neuman E. Zalvide J. Bernards R. DeCaprio J.A. Ewen M.E. Nature. 1997; 386: 177-181Crossref PubMed Scopus (319) Google Scholar, 2Mittnacht S. Paterson H. Olson M.F. Marshall C.J. Curr. Biol. 1997; 7: 219-221Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Winston J.T. Coats S.R. Wang Y.Z. Pledger W.J. Oncogene. 1996; 12: 127-134PubMed Google Scholar, 4Liu J.J. Chao J.R. Jiang M.C., Ng, S.Y. Yen J.-Y. Yang-Yen H.F. Mol. Cell. Biol. 1995; 15: 3654-3663Crossref PubMed Scopus (263) Google Scholar, 5Leone G. DeGregori J. Sears R. Jakoi L. Nevins J.R. Nature. 1997; 387: 422-426Crossref PubMed Scopus (396) Google Scholar). G1 CDKs phosphorylate the three members of the retinoblastoma family of pocket proteins, pRB, p107, and p130, resulting in cell cycle-dependent inactivation of their growth suppressor activities (6Buchkovich K. Duffy L.A. Harlow E. Cell. 1989; 58: 1097-1105Abstract Full Text PDF PubMed Scopus (794) Google Scholar, 7Chen P.L. Scully P. Shew J.Y. Wang J.Y. Lee W.H. Cell. 1989; 58: 1193-1198Abstract Full Text PDF PubMed Scopus (788) Google Scholar, 8DeCaprio J.A. Ludlow J.W. Lynch D. Furukawa Y. Griffin J. Piwnica-Worms H. Huang C.M. Livingston D.M. Cell. 1989; 58: 1085-1095Abstract Full Text PDF PubMed Scopus (687) Google Scholar, 9Mihara K. Cao X.R. Yen A. Chandler S. Driscoll B. Murphree A.L. T'Ang A. Fung Y.K. Science. 1989; 246: 1300-1303Crossref PubMed Scopus (429) Google Scholar, 10Beijersbergen R.L. Carlee L. Kerkhoven R.M. Bernards R. Genes Dev. 1995; 9: 1340-1353Crossref PubMed Scopus (234) Google Scholar, 11Xiao 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, 12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 13Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (868) Google Scholar) (reviewed in Ref. 14Graña X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar). Ectopic expression of cyclin D1 and cyclin E in primary or immortal, nontransformed mammalian fibroblasts shortens the G1 phase of the cell cycle (15Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.Y. Bar S.D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (978) Google Scholar, 16Resnitzky D. Gossen M. Bujard H. Reed S.I. Mol. Cell. Biol. 1994; 14: 1669-1679Crossref PubMed Scopus (987) Google Scholar, 17Ohtsubo M. Roberts J.M. Science. 1993; 259: 1908-1912Crossref PubMed Scopus (659) Google Scholar). The relatively modest effects of ectopic expression of G1 cyclins in primary or immortal, nontransformed mammalian fibroblasts are probably due to a requirement for additional events to ensure full activation of these complexes. Whereas cyclins are limiting subunits for activation of their corresponding CDKs, full activation of cyclin-CDK complexes requires other events also dependent upon mitogenic stimulation (reviewed in Refs. 18Graña X. Reddy E.P. Oncogene. 1995; 11: 211-219PubMed Google Scholar, 19Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2916) Google Scholar, 20Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5072) Google Scholar). In agreement with this idea, microinjection of purified recombinant active cyclin D1-CDK4 or cyclin E-CDK2 complexes in human primary lung fibroblasts bypasses the requirement for mitogenic signaling (21Connell-Crowley L. Elledge S.J. Harper J.W. Curr. Biol. 1998; 8: 65-68Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). It has been suggested that cyclin D1-CDK effects trigger pRB inactivation and activation of E2F-dependent genes including cyclin E (21Connell-Crowley L. Elledge S.J. Harper J.W. Curr. Biol. 1998; 8: 65-68Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), whereas cyclin E-CDK2 effects might be independent of E2F (22Lukas J. Herzinger T. Hansen K. Moroni M.C. Resnitzky D. Helin K. Reed S.I. Bartek J. Genes Dev. 1997; 11: 1479-1492Crossref PubMed Scopus (322) Google Scholar, 23Leng X. Connell-Crowley L. Goodrich D. Harper J.W. Curr. Biol. 1997; 7: 709-712Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Initial studies suggested that pRB was the only substrate of D-type cyclin-CDKs required for a p16-mediated G1 arrest (24Lukas J. Parry D. Aagaard L. Mann D.J. Bartkova J. Strauss M. Peters G. Bartek J. Nature. 1995; 375: 503-506Crossref PubMed Scopus (864) Google Scholar, 25Koh J. Enders G.H. Dynlacht B.D. Harlow E. Nature. 1995; 375: 506-510Crossref PubMed Scopus (521) Google Scholar, 26Medema R.H. Herrera R.E. Lam F. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6289-6293Crossref PubMed Scopus (407) Google Scholar). However, more recent studies demonstrated that, similar to the lack of pRB, lack of both p130 and p107 also prevents a p16-mediated G1 arrest in primary mouse embryo fibroblasts, strongly suggesting that the three pocket proteins are important substrates for the cell cycle-regulatory functions of D-type cyclin-CDKs. Supporting this hypothesis further, it has been shown that both p130 and p107 are phosphorylated in vivo by d-type cyclin-CDK complexes at specific residues, which are not phosphorylated by cyclin E-CDK2 complexes (27Hansen K. Farkas T. Lukas J. Holm K. Ronnstrand L. Bartek J. EMBO J. 2001; 20: 422-432Crossref PubMed Scopus (86) Google Scholar, 28Leng X. Noble M. Adams P.D. Qin J. Harper J.W. Mol. Cell. Biol. 2002; 22: 2242-2254Crossref PubMed Scopus (70) Google Scholar). Phosphorylation of these sites modulates the growth suppressor activities of p130 and p107. It is likely that two parallel pathways, one controlled by pRB and the other by p130/p107, regulate distinct downstream events required for G1progression into S phase. In agreement with this possibility, different sets of E2F-dependent genes are deregulated in mouse embryo fibroblasts lacking pRB and mouse embryo fibroblasts lacking both p130 and p107 (29Hurford R.K. Cobrinik D. Lee M.H. Dyson N. Genes Dev. 1997; 11: 1447-1463Crossref PubMed Scopus (380) Google Scholar). It is currently thought that pRB interacts with E2F1, E2F2, E2F3, and E2F4, whereas p130 and p107 interact with E2F4 but not with E2F1–3 (reviewed in Refs. 14Graña X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar and 30Mayol X. Graña X. Front. Biosci. 1998; 3: 11-24PubMed Google Scholar). The unique ability of pRB to specifically interact with E2F1–3 supports the hypothesis that pRB controls a pathway, which is different from the pathway controlled by p130/p107. Although it seems clear that cells will synthesize DNA when a certain threshold of cyclin D-CDK or cyclin E-CDK2 activity is induced in a cell, the concerted, as well as individual, effects of G1cyclin-CDK activities on the coordinated phosphorylation of the three endogenous pocket proteins and the subsequent effects on E2F-dependent gene expression are not well understood. To address these questions in more detail, we have studied the downstream events induced by both cyclin D1 and cyclin E and their dependence on each other in human T98G and Rat-1 cells. Both cyclins are sufficient to induce mitogenic independent growth. The effects of both cyclins in endogenous pocket protein phosphorylation are clearly distinct. Phosphorylation of certain pocket proteins is sufficient to disrupt, at least partially, a subset of pocket protein-E2F complexes. However, because of the cell cycle-coordinated expression of endogenous members of the pocket protein and E2F families, new pocket protein-E2F complexes are formed even in the presence of active G1CDKs. Surprisingly, we have found that both p130 and p107 specifically interact with E2F1 in a cell cycle-dependent manner. This work demonstrates that the interactions between pocket proteins and E2F family members are more complex than hereto anticipated and suggests cross-talk between these pathways. T98G and Rat-1 cells were maintained in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (FBS) (Sigma) at 37 °C in a humidified atmosphere with 5% CO2. Cells were synchronized in G0 phase by contact inhibition followed by serum starvation. Briefly, cells were grown and kept overconfluent for 2 days. Cells were then trypsinized, counted, and seeded at 2 × 106 cells/plate in 100-mm dishes in MCDB-105 medium without FBS (Sigma). After 12 h, medium was removed, and fresh Dulbecco's modified Eagle's medium without FBS was added. Cells were kept for another 60 h before starting experiments. Roscovitine was used to specifically inhibit CDK2 activity in cells infected with cyclin D1 adenoviruses. Infections were performed in the presence of either the CDK2/CDC2 inhibitor roscovitine (31Meijer L. Borgne A. Mulner O. Chong J.P.J. Blow J.J. Inagaki N. Inagaki M. Delcros J.G. Moulinoux J.P. Eur. J. Biochemistry. 1997; 243: 527-536Crossref PubMed Scopus (1181) Google Scholar) at a concentration of 25 μm, or vehicle (Me2SO). For G2/M blockage and release experiments, serum-starved T98G cells restimulated with 10% FBS or infected with the indicated adenovirus for 22 h were incubated in the presence or absence of nocodazole (10 μm) for an additional 22 h. When indicated, G2/M-synchronized cells were shaken off as previously described (32Mayol X. Garriga J. Graña X. Oncogene. 1996; 13: 237-246PubMed Google Scholar) and then reseeded in fresh medium. Cells were collected at the time points indicated under “Results” and processed for flow cytometric and/or Western blot analysis. Anti-p107 (sc-318), anti-p21 (sc-397), anti-p27 (sc-528), anti-cyclin A (sc-596), anti-Cdk2 (sc-163), anti-E2F1 (sc-193), and anti-E2F4 (sc-512) rabbit polyclonal antibodies and anti-cyclin D1 (sc-8396), anti-cyclin E (sc-247 and sc-248), and anti-E2F1 KH95 (sc-251) mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p130 monoclonal antibody (R27020) was from Transduction Laboratories. Anti-p16 monoclonal antibody (G175–405) was from Pharmingen. Anti-pRb was a mix of monoclonal antibodies (XZ35, XZ56, XZ61, XZ91, XZ105, XZ121, XZ133, and XZ145), which were provided by E. Harlow. Recombinant adenoviruses encoding cyclin D1 and cyclin E (Ad-Cyc D1 and Ad-Cyc E) were provided by J. Albrecht (33Albrecht J.H. Hansen L.K. Cell Growth Differ. 1999; 10: 397-404PubMed Google Scholar). Adenoviruses encoding p16 (Ad-p16) were provided by J. Fueyo. Adenoviruses encoding p21 (Ad-p21) were provided by W. El-Deiry. Adenoviruses encoding enhanced green fluorescent protein (Ad-EGFP) were a gift of P. Ruiz. Viral stocks were amplified using 293 cells and were purified by using CsCl density gradient centrifugation. Viral titers were determined by plaque assay (34Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar). Titers obtained ranged from 5 × 109 to 5 × 1010 plaque-forming units/ml. Infection conditions and optimal multiplicity of infection were previously determined using adenovirus-carrying reporter genes (lacZ, EGFP). T98G and Rat-1 cells were infected at a multiplicity of infection of 50–100 plaque-forming units/cell and 20 plaque-forming units/cell, respectively, for each adenovirus used. Infections were carried out by direct addition of the necessary volume of adenovirus stock to the medium. Whole protein lysates were obtained essentially as described previously (12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar,35Garriga J. Limon A. Mayol X. Rane S.G. Albrecht J.H. Reddy E.P. Andrés V. Graña X. Biochem. J. 1998; 333: 645-654Crossref PubMed Scopus (70) Google Scholar) by lysing cells in buffer containing 50 mm Tris-Cl (pH 7.4), 5 mm EDTA, 250 mm NaCl, 50 mmNaF, 0.1% Triton X-100, 0.1 mmNa3VO4, 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 4 μg/ml aprotinin, and 4 μg/ml pepstatin (lysis buffer). For cyclin D1 kinase assays and certain immunoprecipitations, we used DIP buffer (36Reed M.F. Liu V.F. Ladha M.H. Ando K. Griffin J.D. Weaver D.T. Ewen M.E. Oncogene. 1998; 17: 2961-2971Crossref PubMed Scopus (17) Google Scholar) (see below). Immunoprecipitations and Western blots were performed as previously described (12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 35Garriga J. Limon A. Mayol X. Rane S.G. Albrecht J.H. Reddy E.P. Andrés V. Graña X. Biochem. J. 1998; 333: 645-654Crossref PubMed Scopus (70) Google Scholar). Briefly, protein extracts (250–500 μg) were incubated for 1 h at 4 °C with specific antibodies, and immunocomplexes were precipitated with 25 μl of Protein A-Sepharose beads (Pierce) for 2 h at 4 °C and washed four times. Complexes were eluted from beads by adding 1.5 × Laemmli sample buffer. Whole cell lysates or immunocomplexes were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp.) in 10 mm CAPS (pH 11) containing 10% methanol. 10 and 12% gels were run to determine the expression of cyclins, p16, p21, p27, and E2F proteins; and 6 and 8% gels to determine the phosphorylation status of pocket proteins. The transferred membranes were probed with specific primary antibodies and the corresponding horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Bands were visualized by incubating the membranes with Enhanced Chemiluminescence reagent (PerkinElmer Life Sciences) and exposing the membranes to x-ray film. CDK2 kinase activity was determined from CDK2, cyclin E, and cyclin A immunopurified complexes as described previously (37Graña X., De Luca A. Sang N., Fu, Y. Claudio P.P. Rosenblatt J. Morgan D.O. Giordano A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3834-3838Crossref PubMed Scopus (199) Google Scholar, 38Garriga J. Segura E. Mayol X. Grubmeyer C. Graña X. Biochem. J. 1996; 320: 983-989Crossref PubMed Scopus (23) Google Scholar). Complexes were immunoprecipitated with specific antibodies from whole protein extracts (40 μg for CDK2 and 200 μg for cyclin E). Kinase assays were performed at 30 °C for 30 min in 20 mm HEPES-Na (pH 7.4), 10 mm magnesium acetate, 1 mm dithiothreitol, 20 μm ATP (10 μCi/reaction), and 1 μg of substrate (histone H1). CycD1 kinase activity was determined as described (36Reed M.F. Liu V.F. Ladha M.H. Ando K. Griffin J.D. Weaver D.T. Ewen M.E. Oncogene. 1998; 17: 2961-2971Crossref PubMed Scopus (17) Google Scholar). Briefly, cells were lysed in DIP buffer (50 mm HEPES, pH 7.2, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 10% glycerol, and 0.1% Tween 20) containing freshly added 1 mm dithiothreitol, 1 mm NaF, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm Na3VO4, 10 mmβ-glycerophosphate, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. Active complexes were immunoprecipitated from 800 μg of cell lysate by using CycD1 monoclonal antibodies, collected with protein A-Sepharose (30 μl of 50% slurry containing beads preequilibrated in DIP with 4% bovine serum albumin), and washed four times with DIP buffer containing fresh inhibitors and two additional times with kinase buffer (50 mm HEPES, pH 7.2, 10 mmMgCl2, 5 mm MnCl2, and freshly added 1 mm dithiothreitol). Kinase assays were performed at 30 °C for 30 min in kinase buffer containing 20 μm ATP (10 μCi/reaction) and 1 μg of substrate (GST-pRb C terminus). In all cases, reactions were stopped by adding 1 volume of 2× Laemmli sample buffer and boiling samples for 10 min. Substrates were resolved in 12% SDS-PAGE, and gels were dried and visualized by autoradiography. All measurements were performed on a FACScan (BD Pharmingen). For DNA content quantification, cells were processed as described earlier (12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar). For the cell proliferation assay, cells were plated (see above) in serum-free medium, and 24 h later cells were washed twice with phosphate-buffered saline and stained with 20 μm carboxymethyl fluorescein diacetate succinyl ester (Molecular Probes, Inc., Eugene, OR) in phosphate-buffered saline for 5 min at room temperature. CFSE incorporation was stopped by washing once with 10% FBS-containing medium and two additional times with serum-free medium. At time points indicated, cells were collected, washed with phosphate-buffered saline, and resuspended in 1% FBS-containing phosphate-buffered saline, and green fluorescence was quantified. Data from 10,000 cells were collected and analyzed using ModFitLT version 2.0 software. We were interested in determining the effects that expression of G1 cyclins have on the phosphorylation status of endogenous pocket proteins and the subsequent effects on pocket protein-E2F complexes, expression of E2F-dependent and independent gene products, and, ultimately, entry and progression throughout the cell cycle. To this end, we chose an immortal human cell line (T98G cells), which has been extensively used in cell cycle studies. We also used the rat embryo fibroblast cell line Rat-1. Both T98G and Rat-1 cells are effectively arrested in a quiescent state by serum starvation, and subsequent mitogenic stimulation leads to synchronous cell cycle entry. To ectopically express G1 cyclins and cyclin kinase inhibitors, we utilized replication-defective recombinant adenoviruses. Under our experimental conditions, virtually all cells are transduced by the recombinant adenoviruses at multiplicity of infection values of 50–100 as estimated by using recombinant adenoviruses encoding the green fluorescent protein and β-galactosidase (data not shown). Initial experiments demonstrated that transduction of serum-starved T98G cells with recombinant adenoviruses encoding cyclins D1 (Ad-Cyc D1) or E (Ad-Cyc E), but not β-galactosidase control adenoviruses (Ad-β-gal), for 48 h was sufficient to induce cell cycle entry in the absence of any mitogenic stimuli as determined by fluorescence-activated cell sorting analysis following propidium iodide staining (data not shown; see below). This was also confirmed using Rat-1 cells. To determine the effects of ectopic expression of cyclin D1 and E alone or combined on cell cycle entry, T98G cells were serum-starved for 2 days and then stimulated with FBS or infected with the indicated recombinant adenoviruses at a multiplicity of infection of 50 (Fig. 1). Cells were harvested at the indicated time points and processed for fluorescence-activated cell sorting and Western blot analysis. The expression of the ectopically expressed cyclins is shown in Fig. 1 A. Cells maintained in the absence of serum did not show changes in cell cycle distribution (Fig. 1 B). Similar results were obtained with cells transduced with the β-galactosidase control adenovirus in the absence of serum (data not shown; see below). As expected, stimulation with 10% FBS led to synchronous cell cycle entry. More than 40% of the cells were in S phase by 22 h, and by 30 h most cells were in the next G1 phase following mitosis (Fig. 1 B). Interestingly, ectopic expression of cyclin D1 and cyclin E individually or together led to synchronous cell cycle entry and progression throughout S phase. Cyclin D1 appeared more potent in inducing cell cycle progression than cyclin E (comparepanels 3 and 4). Moreover, when both cyclins were coexpressed, the kinetics of cell cycle progression were very similar to that induced by cyclin D1 alone (comparepanels 3 and 5). To determine whether the expression of cyclins D1 and E alone or combined was able to induce changes in the patterns of phosphorylation of pocket proteins similar to those changes induced by stimulation with FBS, we resolved protein extracts by 6% SDS-PAGE followed by Western blot analysis with specific antibodies. Fig. 1 C shows that expression of both cyclins appears sufficient to induce hyperphosphorylation of the three pocket proteins (see below). p130-phosphorylated forms found in quiescent T98G cells consist of forms 1 and 2 (12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar, 32Mayol X. Garriga J. Graña X. Oncogene. 1996; 13: 237-246PubMed Google Scholar). Stimulation with FBS triggers phosphorylation of p130 to form 3 in mid-G1 (8–10 h in T98G cells) (Fig. 1 C,panel 2) (12Mayol X. Garriga J. Graña X. Oncogene. 1995; 11: 801-808PubMed Google Scholar). p107 is expressed at low levels, and it is found in its hypophosphorylated form in quiescent cells. p107 becomes hyperphosphorylated and expressed at higher levels concomitantly with p130 hyperphosphorylation. pRB also becomes hyperphosphorylated in mid-G1. (Note that the anti-pRB mixture of monoclonal antibodies used in this experiment had a preference for the hypophosphorylated form; whereas the appearance of hyperphosphorylated forms is clear, the change in intensity of the different forms does not reflect a change in the expression of pRB.) Ectopic expression of cyclin D1 in the absence of FBS was apparently sufficient to induce the same changes in protein phosphorylation induced by serum stimulation (Fig. 1 C, comparepanels 2 and 3). Of note, cyclin E appeared less effective in inducing changes in pocket protein phosphorylation and cell cycle progression. Finally, coexpression of cyclins D1 and E exhibited the same effects of cyclin D1 alone. The results of this experiment demonstrate that ectopic expression of either cyclin D1 or cyclin E alone is sufficient to bypass the growth factor requirements necessary to trigger hyperphosphorylation of pocket proteins and cell cycle entry in serum-starved quiescent T98G cells. However, because activation of cyclin D1-CDK complexes is likely to lead to activation of endogenous cyclin E-CDK2 complexes and vice versa, we could not discern the individual effects of each cyclin on pocket protein phosphorylation and cell cycle progression. Similar experiments were also performed using Rat-1 cells. The expression of either cyclin D1 or cyclin E was sufficient to trigger phosphorylation of pocket proteins and cell cycle progression in the absence of serum (data not shown). A strategy to induce activation of cyclin D1-CDK complexes in the absence of CDK2 activity or activation of cyclin E-CDK2 complexes in the absence of D-type cyclin-CDK activity was designed. Our strategy consists of transducing serum-starved T98G cells with recombinant adenoviruses expressing either p16 (an inhibitor of D-type cyclin-CDK activity) (39Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3340) Google Scholar), p21 (an assembly factor for D-type cyclin-CDK complexes and a potent inhibitor of cyclin E-CDK2 complexes) (reviewed in Ref. 20Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5072) Google Scholar), or Ad-β-gal (control). 5 h after transduction, (a) the cells infected with Ad-p16 were infected with Ad-Cyc E, (b) the cells infected with Ad-p21 were infected with Ad-Cyc D1, and (c) the cells infected with Ad-β-gal were infected with Ad-Cyc D1, Ad-Cyc E, or Ad-β-gal. These combinations were predicted to generate both individual and cooperative G1 CDK activities in vivo. To determine whether this was the case, cells were harvested at the indicated time points after transduction with the second group of recombinant adenoviruses (Fig. 2). Whole cell protein lysates were obtained and used to perform kinase assays. Fig. 2 A shows efficient expression of the ectopically expressed proteins, as determined by Western blot analysis. Under the shown exposure times, endogenous cyclin D1 is not detected (see Fig. 1). We determined the kinase activities associated with cyclin D1, cyclin E, and CDK2 by immunoprecipitating the kinase complexes with specific antibodies and performing kinase assays with the immunoprecipitates using the C-terminal domain of pRB and histone H1 as exogenous substrates as indicated in Fig. 2 B. As expected, stimulation of T98G cells with FBS led to up-regulation of cyclin D1-, cyclin E-, and CDK2-associated kinase activities (lanes 1–3). Ectopic expression of cyclin D1 resulted in high levels of cyclin D1-associated kinase activity, which was followed by induction of cyclin E- and CDK2-associated kinase activities (lanes 10 and 11). The differences between cyclin E- and CDK2-associated kinase activities are that the latter consists of cyclin E-CDK2 and cyclin A-CDK2 activities. Of note, coexpression of cyclin D1 and p21 also leads to induction of cyclin D1-associated kinase activity to levels comparable with those induced by serum stimulation (compare lanes 2 and3 with lanes 12 and 13). However, cyclin D1 kinase activity was lower in cells coexpressing cyclin D1 and p21 than in cells expressing cyclin D1 alone (comparelanes 10 and 11 with lanes 12 and 13). This suggests that p21, when expressed at high levels, inhibits cyclin D1-CDK activity (20Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5072) Google Scholar). Importantly, cyclin D1 activation in cells coexpressing p21 is not accompanied by activation of cyclin E or CDK2-associated kinase activities (compare lanes 10 and 11with lanes 12 and 13). Thus, cotransfection of cyclin D1 and p21 results in individual activation of cyclin D1-CDK complexes. An independent strategy to activate D-type cyclin-CDK activity in the absence of cyclin E-CDK2 activity consisted of treating cells expressing exogenous cyclin D1 with roscovitine, an inhibitor of CDK2/CDC2 (see below). On the other hand, expression of cyclin E leads to induction of cyclin E and CDK2-associated kinase activities and, to a lesser extent, cyclin D1-associated kinase activity (compare lanes 6and 7 with lanes 4 and 5). Notably, coexpression of cyclin E and p16" @default.
• W2081736214 created "2016-06-24" @default.
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• W2081736214 creator A5030400129 @default.
• W2081736214 creator A5031949375 @default.
• W2081736214 creator A5064541586 @default.
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• W2081736214 date "2002-12-01" @default.
• W2081736214 modified "2023-09-26" @default.
• W2081736214 title "G1 Cyclin/Cyclin-dependent Kinase-coordinated Phosphorylation of Endogenous Pocket Proteins Differentially Regulates Their Interactions with E2F4 and E2F1 and Gene Expression" @default.
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