THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 11, Issue of March 14, pp. 9754–9760, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Cyclin D1-dependent Kinase Associates with the Pre-replication Complex and Modulates RB⅐MCM7 Binding* Received for publication, November 27, 2002, and in revised form, December 23, 2002 Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212088200 Andrew B. Gladden and J. Alan Diehl‡ From the Leonard and Madlyn Abramson Family Cancer Research Institute, Department of Cancer Biology, Abramson Family Cancer Center of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 The capacity of the cyclin D-dependent kinase to pro- Following ORC binding, CDC6 (5) and Cdt1 (6) associate and ⅐ mote G1 progression through modulation of RB E2F is function to recruit the minichromosome maintenance proteins well documented. We now demonstrate that the cyclin (MCM), a group of highly conserved proteins essential for the D1/CDK4 kinase binds to components of the MCM com- initiation of DNA synthesis (7–9). The MCM complex is com- Downloaded from plex. MCM7 and MCM3 were identified as cyclin D1- posed of six distinct, but related polypeptides, MCM2, MCM3, binding proteins. Catalytically active cyclin D1/CDK4 MCM4, MCM5, MCM6, and MCM7 (10), that form a hexameric complexes were incorporated into chromatin-bound helicase the activity of which is essential for initiation and protein complexes with the same kinetics as MCM7 and elongation of the replication fork (11). Origin firing at the G1/S MCM3, where they associated specifically with MCM7. boundary is also highly regulated, but the precise regulatory Although the cyclin D1-dependent kinase did not phos- mechanisms remain unclear. Both cyclin E/CDK2 and a dis- www.jbc.org phorylate MCM7, active cyclin D1/CDK4, but not cyclin tinct kinase composed of Cdc7 and Dbf4 function to promote S E/CDK2, did catalyze the dissociation of an RB⅐MCM7 phase entry, perhaps through direct phosphorylation of MCM complex. Finally, expression of an active D1/CDK4 ki- subunits (12–16). nase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data sug- Although current data support the notion that the phospho- at University of Pennsylvania Library on September 24, 2008 gest that D1/CDK4 complexes play a direct role in alter- rylation status of pre-RC subunits determines activity, it is also ing an inhibitory RB⅐MCM7 complex possibly allowing becoming clear that interactions with cell cycle inhibitors also for setting of the origin in preparation for DNA contribute. The RB tumor suppressor protein functions as the replication. primary block that prevents premature activation of cell cycle entry and thus premature S phase entry. Although the cell cycle inhibitory activity of RB is most thoroughly understood in External stimuli activate cellular signaling cascades, terms of its ability to directly repress E2F-dependent transcrip- thereby promoting the induction of cellular activities needed to tion (17), RB also directly associates with the pre-RC through initiate DNA replication. During the G1 phase, growth factor- MCM7 (18) and perhaps subunits of ORC (19). initiated signals promote the accumulation and assembly of the Given that RB associates with components of the pre-RC and D-type cyclins (D1, D2, D3) with their cognate cyclin-dependent that RB is the only known substrate of cyclin D1, we considered 1 kinase (CDK4 or 6). The active holoenzyme promotes G1 phase the possibility that cyclin D1 might target components of the progression and S phase entry by virtue of its ability to phos- DNA replication machinery that are inhibited by RB. Using phorylate the retinoblastoma protein (RB) and titrate the CDK cyclin D1 as bait for a yeast two-hybrid screen, we identified inhibitors p27Kip1 and p21Cip1 (1, 2). Initial phosphorylation of MCM7 as a cyclin D1 binding partner. We demonstrate that RB and titration of the CDK inhibitors expedites the activation cyclin D1 mediates binding of the cyclin D1/CDK4 complex to of the cyclin E/CDK2 kinase, thereby completing RB phospho- MCM7 in mammalian cells. We demonstrate that the cyclin rylation and inactivation prior to initiation of DNA replication. D1/CDK4 kinase is incorporated into chromatin-bound protein Initiation of DNA replication at the G1/S phase boundary is complexes, with the same kinetics as MCM7 and that the cyclin meticulously regulated to ensure the cell has assembled the D1/CDK4 kinase specifically dissociates RB⅐MCM7 complexes, appropriate machinery needed for high fidelity genome dupli- thereby facilitating establishment of the pre-RC. cation. Prior to S phase entry, pre-replication complexes (pre- EXPERIMENTAL PROCEDURES RC) form at replication origins during G1 phase. Origins are Yeast Two-hybrid Screen—Yeast strain Y190 (his3 leu2 trp1), which first marked by the origin recognition complex (ORC), which is carries GAL4-dependent lacZ and HIS3 genes, was transformed with composed of six subunits (ORC1–6) (3) and serves as a landing pAS2D1, and TRPϩ transformants were examined for lacZ and HIS3 pad for recruitment of the DNA replication machinery (4). expression via a ␤-galactosidase assay and growth on media lacking histidine. A lacZϪ HIS3Ϫ TRP1ϩ clone was transformed with a HEK293 cDNA library (Clontech) fused to the GAL4 activation domain in plas- * This work was supported by National Institutes of Health Grant mid pGAD-GH, a LEU2 yeast plasmid. Transformants were replica CA93237 (to J. A. D.). The costs of publication of this article were plated onto indicator plates to score for growth in media lacking histi- defrayed in part by the payment of page charges. This article must dine and ␤-galactosidase activity. This selection resulted in 51 positive therefore be hereby marked “advertisement” in accordance with 18 colonies. Further analysis revealed that the cDNAs encoded by each of U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. E-mail: adiehl@ the 51 colonies interacted with GAL4-D1 and not other bait constructs mail.med.upenn.edu. containing GAL4 fusions with p53 or large T antigen. 1 The abbreviations used are: CDK, cyclin-dependent kinase; MEF, Cell Culture Conditions and Transfections—NIH-3T3 cells and murine embryonic fibroblast; MCM, minichromosome maintenance; RBϪ/Ϫ mouse embryonic fibroblasts (MEFs) were maintained in Dul- ORC, origin recognition complex; RB, retinoblastoma protein; Pipes, becco’s modified Eagle’s medium containing glutamine supplemented 1,4-piperazinediethanesulfonic acid; pre-RC, pre-replication complex; with antibiotics (BioWhittaker) and 10% fetal calf serum (BioWhit- HA, hemagglutinin; IP, immunoprecipitation; HRP, horseradish perox- taker). Derivatives of NIH-3T3 cells engineered to overexpress Flag- idase; GST, glutathione S-transferase. tagged cyclin D1 and Flag-tagged cyclin D1-T286A have been described 9754 This paper is available on line at http://www.jbc.org Cyclin D1/CDK4-dependent Dissociation of a RB⅐MCM7 Complex 9755 previously (20). Insect Sf9 cells were grown in Grace’s medium supple- mented with 10% heat-inactivated fetal calf serum. Procedures for manipulation of baculoviruses have been described previously (21). Transient expression of plasmids encoding HA-tagged MCM7, Flag- tagged cyclin D1, CDK4, RB, CDK4/K35M, Myc-tagged cyclin E, and CDK2 was achieved by using LipofectAMINE Plus (Invitrogen) accord- ing to the instructions from the manufacturer. Purification of Active Cyclin-CDK Complexes—Following infection of insect Sf9 cells with the indicated viral supernatants, cells were lysed in Tween 20 buffer (50 mM HEPES (pH 8), 150 mM NaCl, 2.5 mM EGTA, 1mM EDTA, 0.1% Tween 20) and cleared by sedimentation for 10 min. Flag-D1 complexes were purified by affinity chromatography using M2-agarose (Sigma) and eluted with FLAG peptide (5 ␮g/ml) (Sigma) FIG.1. Cyclin D1 binds to the COOH terminus of MCM7. A, predicted amino acid sequence, corresponding to residues 587–719, of solubilized in kinase buffer (50 mM HEPES (pH 8), 10 mM MgCl , 2.5 2 the MCM7 cDNA identified in a cyclin D1 yeast two-hybrid screen. B, mM EGTA, 1 mM dithiothreitol, 20 ␮M ATP, 10 mM ␤-glycerol phos- cyclin D1 binds specifically to MCM7 in vitro. MCM2–7 were labeled phate, 0.1 mM NaVO ,and1mM NaF). Cyclin E/His-CDK2 complexes 3 with [35S]methionine by in vitro transcription and translation. Labeled were purified using TALON metal affinity resin (Clontech) and eluted proteins were mixed with Sf9 lysates programmed with Flag-D1 and with 150 mM imidazole solubilized in His-CDK2 kinase buffer (50 mM immunoprecipitated with control rabbit serum (NRS) or with the M2 ␮ HEPES (pH 8), 10 mM MgCl2,1mM dithiothreitol, 20 M ATP, 10 mM monoclonal antibody. Ten percent of the MCM proteins mixed with ␤ Downloaded from -glycerol phosphate, 0.1 mM NaVO3,and1mM NaF). Purified com- Flag-D1 were resolved by SDS-PAGE (lower panel). MCM proteins were plexes were either used immediately or stored at 4 °C. visualized by autoradiography. Chromatin-binding Assays—Cells harvested by trypsinization were lysed in CSKϩ buffer (10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM Myc-tagged cyclin E was visualized with the 9E10 monoclonal antibody sucrose, 3 mM MgCl , 0.5% Triton X-100, 1 mM ATP, 0.1 mM phenyl- 2 followed by biotinylated anti-mouse and subsequently streptavidin methylsulfonyl fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml Texas Red. MCM7 was visualized using a polyclonal rabbit anti-MCM7 pepstatin, and 25 mM ␤-glycerol phosphate). Lysates were incubated on (PharMingen) followed by fluorescein isothiocyanate-conjugated anti- ice for 20 min and then subjected to low speed centrifugation (1000 ϫ g) www.jbc.org rabbit (Amersham Biosciences). RB was visualized using a polyclonal for 5 min. The soluble fraction was removed, and the chromatin pellet rabbit anti-RB (Santa Cruz Biotechnologies, sc-50-G) followed by fluo- was washed one time with DNase I buffer and then digested with rescein isothiocyanate-conjugated anti-rabbit.