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• W2952456576 abstract "•Cyclin D, but not other cyclins, targets a C-terminal alpha-helix docking motif on Rb•Helix-based docking is shared by the p107 and p130 Rb-family members across metazoans•Helix-based docking is a major driver of Rb phosphorylation and the G1/S transition The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb’s tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms. The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb’s tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms. The cyclin-dependent kinases Cdk4 and Cdk6 (Cdk4,6) are activated by the D-type cyclins D1, D2, and D3 (cyclin D) to drive cell-cycle progression from G1 to S phase (Morgan, 1997Morgan D.O. Cyclin-dependent kinases: engines, clocks, and microprocessors.Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1802) Google Scholar, Sherr and Roberts, 2004Sherr C.J. Roberts J.M. Living with or without cyclins and cyclin-dependent kinases.Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (908) Google Scholar). One important target of Cdk4,6 is the retinoblastoma tumor suppressor protein Rb, which binds and inhibits the activating E2F transcription factors. Rb phosphorylation promotes its dissociation from E2Fs and thereby drives the expression of E2F-target genes that initiate DNA replication (Bertoli et al., 2013Bertoli C. Skotheim J.M. de Bruin R.A.M. Control of cell cycle transcription during G1 and S phases.Nat. Rev. Mol. Cell Biol. 2013; 14: 518-528Crossref PubMed Scopus (880) Google Scholar, Dick and Rubin, 2013Dick F.A. Rubin S.M. Molecular mechanisms underlying RB protein function.Nat. Rev. Mol. Cell Biol. 2013; 14: 297-306Crossref PubMed Scopus (374) Google Scholar, Sherr and McCormick, 2002Sherr C.J. McCormick F. The RB and p53 pathways in cancer.Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1312) Google Scholar). The importance of Rb phosphorylation and the frequent observation of increased Cdk4,6 activity in cancer has contributed to the consensus model that these kinases, activated by their D-type cyclin partners, phosphorylate and inhibit Rb to drive cell-cycle progression (Burkhart and Sage, 2008Burkhart D.L. Sage J. Cellular mechanisms of tumour suppression by the retinoblastoma gene.Nat. Rev. Cancer. 2008; 8: 671-682Crossref PubMed Scopus (688) Google Scholar, Lundberg and Weinberg, 1998Lundberg A.S. Weinberg R.A. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes.Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (856) Google Scholar). In the current model, cyclin D-Cdk4,6 activity gradually increases until it triggers a positive feedback loop that commits cells to passing the restriction point just prior to the G1/S transition (Merrick et al., 2011Merrick K.A. Wohlbold L. Zhang C. Allen J.J. Horiuchi D. Huskey N.E. Goga A. Shokat K.M. Fisher R.P. Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation.Mol. Cell. 2011; 42: 624-636Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, Pardee, 1974Pardee A.B. A restriction point for control of normal animal cell proliferation.Proc. Natl. Acad. Sci. USA. 1974; 71: 1286-1290Crossref PubMed Scopus (1061) Google Scholar, Schwarz et al., 2018Schwarz C. Johnson A. Kõivomägi M. Zatulovskiy E. Kravitz C.J. Doncic A. Skotheim J.M. A precise Cdk activity threshold determines passage through the restriction point.Mol. Cell. 2018; 69: 253-264Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). As cells progress through G1, cyclin D-Cdk4,6 gradually phosphorylates Rb and triggers the onset of E2F-dependent expression of cyclins E and A (Bertoli et al., 2013Bertoli C. Skotheim J.M. de Bruin R.A.M. Control of cell cycle transcription during G1 and S phases.Nat. Rev. Mol. Cell Biol. 2013; 14: 518-528Crossref PubMed Scopus (880) Google Scholar). Cyclins E and A then bind Cdk1 and Cdk2 to form complexes that continue to phosphorylate Rb (Merrick et al., 2011Merrick K.A. Wohlbold L. Zhang C. Allen J.J. Horiuchi D. Huskey N.E. Goga A. Shokat K.M. Fisher R.P. Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation.Mol. Cell. 2011; 42: 624-636Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, Morgan, 2007Morgan D.O. The Cell Cycle. New Science Press, 2007Google Scholar, Narasimha et al., 2014Narasimha A.M. Kaulich M. Shapiro G.S. Choi Y.J. Sicinski P. Dowdy S.F. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.eLife. 2014; 3: 1068Crossref Google Scholar). In addition, cyclin E- and A-dependent Cdk complexes phosphorylate and inhibit the E3 ubiquitin ligase APC/C activating subunit Cdh1, stabilizing APC/CCdh1 substrates, including cyclin A (Di Fiore et al., 2015Di Fiore B. Davey N.E. Hagting A. Izawa D. Mansfeld J. Gibson T.J. Pines J. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.Dev. Cell. 2015; 32: 358-372Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Jaspersen et al., 1999Jaspersen S.L. Charles J.F. Morgan D.O. Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14.Curr. Biol. 1999; 9: 227-236Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, Kramer et al., 2000Kramer E.R. Scheuringer N. Podtelejnikov A.V. Mann M. Peters J.M. Mitotic regulation of the APC activator proteins CDC20 and CDH1.Mol. Biol. Cell. 2000; 11: 1555-1569Crossref PubMed Scopus (360) Google Scholar, Zachariae et al., 1998Zachariae W. Schwab M. Nasmyth K. Seufert W. Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.Science. 1998; 282: 1721-1724Crossref PubMed Scopus (442) Google Scholar). The sequential activation of these interconnected positive feedback loops progressively drives commitment to cell division in the face of exposure to anti-proliferative conditions (Cappell et al., 2016Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, Cappell et al., 2018Cappell S.D. Mark K.G. Garbett D. Pack L.R. Rape M. Meyer T. EMI1 switches from being a substrate to an inhibitor of APC/CCDH1 to start the cell cycle.Nature. 2018; 558: 313-317Crossref PubMed Scopus (65) Google Scholar, Pardee, 1974Pardee A.B. A restriction point for control of normal animal cell proliferation.Proc. Natl. Acad. Sci. USA. 1974; 71: 1286-1290Crossref PubMed Scopus (1061) Google Scholar, Schwarz et al., 2018Schwarz C. Johnson A. Kõivomägi M. Zatulovskiy E. Kravitz C.J. Doncic A. Skotheim J.M. A precise Cdk activity threshold determines passage through the restriction point.Mol. Cell. 2018; 69: 253-264Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Yung et al., 2007Yung Y. Walker J.L. Roberts J.M. Assoian R.K. A Skp2 autoinduction loop and restriction point control.J. Cell Biol. 2007; 178: 741-747Crossref PubMed Scopus (41) Google Scholar, Zetterberg and Larsson, 1985Zetterberg A. Larsson O. Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells.Proc. Natl. Acad. Sci. USA. 1985; 82: 5365-5369Crossref PubMed Scopus (288) Google Scholar). However, in opposition to the prevailing model of gradually increasing cyclin D-Cdk4,6 activity triggering G1/S, cyclin D levels are nearly constant through G1 (Hitomi and Stacey, 1999Hitomi M. Stacey D.W. Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.Curr. Biol. 1999; 9: 1075-1084Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Moreover, Rb is mono-phosphorylated during early- to mid-G1, suggesting that cyclin D-Cdk4,6 activity does not gradually increase through G1 (Narasimha et al., 2014Narasimha A.M. Kaulich M. Shapiro G.S. Choi Y.J. Sicinski P. Dowdy S.F. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.eLife. 2014; 3: 1068Crossref Google Scholar). E2F-dependent transcription increases at the same time that Cdk2 activity increases in late G1, implying that cyclin D-dependent mono-phosphorylated Rb is still capable of interacting with E2F transcription factors to inhibit transcription. This raises the possibility that Rb inactivation in late G1 is due to hyper-phosphorylation by Cdk2 kinase complexes, and cyclin D-Cdk4,6 promotes the G1/S transition through a different mechanism (Narasimha et al., 2014Narasimha A.M. Kaulich M. Shapiro G.S. Choi Y.J. Sicinski P. Dowdy S.F. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.eLife. 2014; 3: 1068Crossref Google Scholar). If not Rb, what could be the main target of cyclin D-Cdk4,6 driving cell-cycle progression? Possible substrates, whose phosphorylation promotes cell-cycle progression, include a mediator of antiproliferative transforming growth factor β (TGF-β) signaling Smad3 (Matsuura et al., 2004Matsuura I. Denissova N.G. Wang G. He D. Long J. Liu F. Cyclin-dependent kinases regulate the antiproliferative function of Smads.Nature. 2004; 430: 226-231Crossref PubMed Scopus (416) Google Scholar), an APC/C co-activator Cdh1 (The et al., 2015The I. Ruijtenberg S. Bouchet B.P. Cristobal A. Prinsen M.B.W. van Mourik T. Koreth J. Xu H. Heck A.J.R. Akhmanova A. et al.Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.Nat. Commun. 2015; 6: 5906Crossref PubMed Scopus (44) Google Scholar), and a cell-cycle transcription factor FOXM1 (Anders et al., 2011Anders L. Ke N. Hydbring P. Choi Y.J. Widlund H.R. Chick J.M. Zhai H. Vidal M. Gygi S.P. Braun P. Sicinski P. A systematic screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in cancer cells.Cancer Cell. 2011; 20: 620-634Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Further supporting an oncogenic role for Cdk4,6, there is a growing body of literature showing that Cdks directly phosphorylate metabolic enzymes to regulate metabolism in yeast and human cells (Ewald, 2018Ewald J.C. How yeast coordinates metabolism, growth and division.Curr. Opin. Microbiol. 2018; 45: 1-7Crossref Scopus (25) Google Scholar, Ewald et al., 2016Ewald J.C. Kuehne A. Zamboni N. Skotheim J.M. The yeast cyclin-dependent kinase routes carbon fluxes to fuel cell cycle progression.Mol. Cell. 2016; 62: 532-545Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, Salazar-Roa and Malumbres, 2017Salazar-Roa M. Malumbres M. Fueling the cell division cycle.Trends Cell Biol. 2017; 27: 69-81Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Zhao et al., 2016Zhao G. Chen Y. Carey L. Futcher B. Cyclin-dependent kinase co-ordinates carbohydrate metabolism and cell cycle in S. cerevisiae.Mol. Cell. 2016; 62: 546-557Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). More specifically, in mammalian cells, cyclin D3-Cdk6 kinase complexes phosphorylate and inactivate the key glycolytic enzymes PFKP and PKM2 to shunt glycolytic intermediates toward NADPH and GSH production, which mitigates ROS accumulation to promote cell survival (Wang et al., 2017Wang H. Nicolay B.N. Chick J.M. Gao X. Geng Y. Ren H. Gao H. Yang G. Williams J.A. Suski J.M. et al.The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival.Nature. 2017; 546: 426-430Crossref PubMed Scopus (200) Google Scholar). These Rb-independent roles for cyclin D-Cdk4,6 in promoting cell proliferation raise the question as to what is the in vivo function of the cyclin D-Cdk4,6-Rb interaction. To determine the function of Rb phosphorylation by cyclin D-Cdk4,6, we sought to generate variants of Rb that could no longer interact with cyclin D-Cdk4,6 while preserving all the other interactions with other cyclin-Cdk complexes. The specificity of substrate binding and phosphorylation by cyclin-Cdk complexes is generally determined by the ability of the cyclin to recognize docking sites on substrates (Morgan, 2007Morgan D.O. The Cell Cycle. New Science Press, 2007Google Scholar). Previously identified docking sites on substrates are short linear amino acid motifs. In budding yeast, the G1 cyclin Cln2 recognizes an LP docking motif and the S phase cyclins Clb5 and Clb3 recognize substrates with RxL docking motifs (Bhaduri and Pryciak, 2011Bhaduri S. Pryciak P.M. Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes.Curr. Biol. 2011; 21: 1615-1623Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Cross and Jacobson, 2000Cross F.R. Jacobson M.D. Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin.Mol. Cell. Biol. 2000; 20: 4782-4790Crossref PubMed Scopus (45) Google Scholar, Kõivomägi et al., 2011Kõivomägi M. Valk E. Venta R. Iofik A. Lepiku M. Morgan D.O. Loog M. Dynamics of Cdk1 substrate specificity during the cell cycle.Mol. Cell. 2011; 42: 610-623Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Loog and Morgan, 2005Loog M. Morgan D.O. Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates.Nature. 2005; 434: 104-108Crossref PubMed Scopus (295) Google Scholar). In animal cells, cyclin A-Cdk2 and cyclin E-Cdk2 complexes also utilize RxL-based docking through their α1 helix hydrophobic patches to bind and phosphorylate substrate proteins including Rb, p107, p27, and Cdc6 (Adams et al., 1999Adams P.D. Li X. Sellers W.R. Baker K.B. Leng X. Harper J.W. Taya Y. Kaelin Jr., W.G. Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.Mol. Cell. Biol. 1999; 19: 1068-1080Crossref PubMed Scopus (162) Google Scholar, Hirschi et al., 2010Hirschi A. Cecchini M. Steinhardt R.C. Schamber M.R. Dick F.A. Rubin S.M. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.Nat. Struct. Mol. Biol. 2010; 17: 1051-1057Crossref PubMed Scopus (87) Google Scholar, Russo et al., 1996Russo A.A. Jeffrey P.D. Patten A.K. Massagué J. Pavletich N.P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex.Nature. 1996; 382: 325-331Crossref PubMed Scopus (799) Google Scholar, Schulman et al., 1998Schulman B.A. Lindstrom D.L. Harlow E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.Proc. Natl. Acad. Sci. USA. 1998; 95: 10453-10458Crossref PubMed Scopus (308) Google Scholar, Takeda et al., 2001Takeda D.Y. Wohlschlegel J.A. Dutta A. A bipartite substrate recognition motif for cyclin-dependent kinases.J. Biol. Chem. 2001; 276: 1993-1997Crossref PubMed Scopus (110) Google Scholar, Wohlschlegel et al., 2001Wohlschlegel J.A. Dwyer B.T. Takeda D.Y. Dutta A. Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclin-dependent kinases.Mol. Cell. Biol. 2001; 21: 4868-4874Crossref PubMed Scopus (67) Google Scholar). While cyclin D appears to have a hydrophobic patch on its α1 helix similar to cyclins E and A, it has not been shown if this patch also recognizes RxL motifs. Cyclin D has an N-terminal LxCxE motif, which binds Rb’s LxCxE cleft (Dick and Rubin, 2013Dick F.A. Rubin S.M. Molecular mechanisms underlying RB protein function.Nat. Rev. Mol. Cell Biol. 2013; 14: 297-306Crossref PubMed Scopus (374) Google Scholar, Dowdy et al., 1993Dowdy S.F. Hinds P.W. Louie K. Reed S.I. Arnold A. Weinberg R.A. Physical interaction of the retinoblastoma protein with human D cyclins.Cell. 1993; 73: 499-511Abstract Full Text PDF PubMed Scopus (691) Google Scholar). However, mutation of this LxCxE cleft has only a modest effect in vivo suggesting that either the cyclin D-Rb interaction is of limited importance or that there exist additional docking interactions. Supporting the existence of an additional cyclin D-Rb docking interaction, truncation of the Rb C terminus disrupts phosphorylation by cyclin D1-Cdk4 in vitro, and this truncated Rb variant slows division and inhibits E2F-dependent gene expression in cultured cells (Gorges et al., 2008Gorges L.L. Lents N.H. Baldassare J.J. The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.Am. J. Physiol. Cell Physiol. 2008; 295: C1151-C1160Crossref PubMed Scopus (12) Google Scholar, Pan et al., 2001Pan W. Cox S. Hoess R.H. Grafström R.H. A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein.Cancer Res. 2001; 61: 2885-2891PubMed Google Scholar, Wallace and Ball, 2004Wallace M. Ball K.L. Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4.Mol. Cell. Biol. 2004; 24: 5606-5619Crossref PubMed Scopus (18) Google Scholar). In this study, we analyzed the docking interactions between Rb and cyclin D-Cdk4,6 complexes. We found that cyclin D-Cdk4,6 targets the Rb family of proteins for phosphorylation primarily by docking a C-terminal alpha-helix. Importantly, this Rb C-terminal helix is not recognized by the other major cell-cycle cyclin-Cdk complexes cyclin E-Cdk2, cyclin A-Cdk2, and cyclin B-Cdk1. Thus, mutation of this helix disrupts cyclin D’s ability to phosphorylate Rb and preserves Rb regulation by other cyclin-Cdk complexes. Disruption of helix-based docking reduced Rb phosphorylation, induced G1 cell-cycle arrest in cell lines, and slowed tumor growth in vivo. Taken together, our results show that cyclin D-Cdk4,6 phosphorylates and inhibits Rb via a C-terminal helix and that this interaction is a major driver of cell proliferation. To determine the function of Rb phosphorylation by cyclin D-Cdk4,6, we sought to generate an Rb variant that does not interact with cyclin D-Cdk4,6, but does interact with other cyclin-Cdk complexes (Figures 1A and 1B ). To identify specific cyclin D-Rb interactions, we performed in vitro kinase assays on Rb protein variants with a panel of purified cyclin-Cdk complexes (Schwarz et al., 2018Schwarz C. Johnson A. Kõivomägi M. Zatulovskiy E. Kravitz C.J. Doncic A. Skotheim J.M. A precise Cdk activity threshold determines passage through the restriction point.Mol. Cell. 2018; 69: 253-264Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Figure S1). In contrast to other studies of Rb phosphorylation that used Rb fragments, we were able to purify full-length Rb protein from bacteria for our study. In these kinase assay experiments, mutation of Rb residues required for docking interactions manifest as reduced kinase activity toward Rb. Compared with other cyclin-Cdk complexes, cyclin D-Cdk4,6 exhibited no detectable kinase activity toward the model Cdk substrate histone H1 (Matsushime et al., 1994Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.Y. D-type cyclin-dependent kinase activity in mammalian cells.Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1025) Google Scholar) but was capable of phosphorylating Rb (Figures 1C–1E). To test the effect of mutating known cyclin D-Rb docking interactions, we first removed the LxCxE binding cleft in the Rb pocket domain that interacts with proteins containing the LxCxE motif, such as viral oncoproteins and cyclin D (Dick et al., 2000Dick F.A. Sailhamer E. Dyson N.J. Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (104) Google Scholar, Dowdy et al., 1993Dowdy S.F. Hinds P.W. Louie K. Reed S.I. Arnold A. Weinberg R.A. Physical interaction of the retinoblastoma protein with human D cyclins.Cell. 1993; 73: 499-511Abstract Full Text PDF PubMed Scopus (691) Google Scholar, Markey et al., 2007Markey M.P. Bergseid J. Bosco E.E. Stengel K. Xu H. Mayhew C.N. Schwemberger S.J. Braden W.A. Jiang Y. Babcock G.F. et al.Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function.Oncogene. 2007; 26: 6307-6318Crossref PubMed Scopus (61) Google Scholar). This resulted in only a 1.7-fold ± 0.3-fold reduction in phosphorylation by cyclin D kinase complexes (Figures 1C and S1L), consistent with reports that the LxCxE-docking interaction alone is weak and its removal has modest effects in cells (Dick et al., 2000Dick F.A. Sailhamer E. Dyson N.J. Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (104) Google Scholar, Guiley et al., 2015Guiley K.Z. Liban T.J. Felthousen J.G. Ramanan P. Litovchick L. Rubin S.M. Structural mechanisms of DREAM complex assembly and regulation.Genes Dev. 2015; 29: 961-974Crossref PubMed Scopus (69) Google Scholar). Moreover, we observed that previously reported LxCxE cleft mutations similarly affected cyclin E-, A-, and B-dependent phosphorylation of Rb in vitro and therefore may not be specific for cyclin D (Figures 1C and S1L). Next, we tested the effect of mutating the RxL motifs on Rb that are reported to interact with the hydrophobic patch region of the S phase cyclins E and A (Adams et al., 1999Adams P.D. Li X. Sellers W.R. Baker K.B. Leng X. Harper J.W. Taya Y. Kaelin Jr., W.G. Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.Mol. Cell. Biol. 1999; 19: 1068-1080Crossref PubMed Scopus (162) Google Scholar, Hirschi et al., 2010Hirschi A. Cecchini M. Steinhardt R.C. Schamber M.R. Dick F.A. Rubin S.M. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.Nat. Struct. Mol. Biol. 2010; 17: 1051-1057Crossref PubMed Scopus (87) Google Scholar, Schulman et al., 1998Schulman B.A. Lindstrom D.L. Harlow E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.Proc. Natl. Acad. Sci. USA. 1998; 95: 10453-10458Crossref PubMed Scopus (308) Google Scholar). We generated an Rb variant lacking all 5 RxL sequences in its unstructured regions (Figure 1A and 1B). Compared to wild type Rb, this RxL variant of Rb was phosphorylated 2.0-fold ± 0.1-fold less by cyclin E-Cdk2 and 2.9-fold ± 0.2-fold less by cyclin A-Cdk2 (Figures 1C and S1L). This implies that, while cyclins E and A use RxL docking as previously reported, this is not the only mechanism they use to identify their substrates. Cyclin B-Cdk1 phosphorylation of Rb was unchanged by mutating the RxL motifs, suggesting that cyclin B does not use its hydrophobic patch to phosphorylate Rb. While it has not been studied extensively, cyclin D has an α1 helix hydrophobic patch as cyclins E, A, and B, but it is composed of different amino acid residues (Morgan, 2007Morgan D.O. The Cell Cycle. New Science Press, 2007Google Scholar). We observed that the Rb variant lacking RxL motifs was phosphorylated 4.1-fold ± 0.4-fold less by cyclin D-Cdk4,6, suggesting that cyclin D recognizes RxL motifs as other cyclins (Figures 1C and S1L). While the RxL docking is clearly important for cyclin D, it is shared with other cyclins so that its mutation would not specifically disrupt the cyclin D-Rb interaction. To generate an Rb protein variant that does not interact with cyclin D-Cdk4,6, but does interact with other cyclin-Cdk complexes, we next examined the effect of truncating the final 37 amino acids of Rb. Truncation of the Rb C terminus, which included these 37 amino acids, was previously shown to reduce Rb phosphorylation in vitro and promote Rb’s ability to arrest cells in G1 (Gorges et al., 2008Gorges L.L. Lents N.H. Baldassare J.J. The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.Am. J. Physiol. Cell Physiol. 2008; 295: C1151-C1160Crossref PubMed Scopus (12) Google Scholar, Pan et al., 2001Pan W. Cox S. Hoess R.H. Grafström R.H. A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein.Cancer Res. 2001; 61: 2885-2891PubMed Google Scholar, Wallace and Ball, 2004Wallace M. Ball K.L. Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4.Mol. Cell. Biol. 2004; 24: 5606-5619Crossref PubMed Scopus (18) Google Scholar) (Figure S2). C-terminal truncation of Rb reduced phosphorylation by cyclin D1-Cdk4,6 20-fold ± 10-fold (Figures 1C and S1L) and increased the Michaelis-Menten constant KM beyond our measurement limit of ∼5 μM (Figure S2G), indicating the presence of a docking interaction in the final 37 amino acids of the Rb C terminus. Importantly, this C-terminal truncation is specific for cyclin D-Cdk4,6 complexes and does not affect the phosphorylation of Rb by cyclin E-Cdk2, cyclin A-Cdk2, and cyclin B-Cdk1 complexes (Figures 1C, 1F, and S2H). We next sought to determine how the Rb C terminus promotes phosphorylation by cyclin D-Cdk4,6. Cyclin substrate docking has previously been shown to arise from short linear motifs of a few amino acids in intrinsically disordered regions on the target proteins, a common mechanism for evolution of protein-protein interactions (Bloom and Cross, 2007Bloom J. Cross F.R. Multiple levels of cyclin specificity in cell-cycle control.Nat. Rev. Mol. Cell Biol. 2007; 8: 149-160Crossref PubMed Scopus (402) Google Scholar, Davey et al., 2015Davey N.E. Cyert M.S. Moses A.M. Short linear motifs - ex nihilo evolution of protein regulation.Cell Commun. Signal. 2015; 13: 43Crossref PubMed Scopus (117) Google Scholar). However, such a short linear motif model is unlikely to explain the interaction of cyclin D with the Rb C terminus because in vitro Rb phosphorylation by cyclin D-Cdk4 is affected by mutations over a large range of amino acids (Wallace and Ball, 2004Wallace M. Ball K.L. Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4.Mol. Cell. Biol. 2004; 24: 5606-5619Crossref PubMed Scopus (18) Google Scholar). While the Rb C terminus is intrinsically disordered, it is known to adopt structure when bound to other proteins (Rubin et al., 2005Rubin S.M. Gall A.-L. Zheng N. Pavletich N.P. Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release.Cell. 2005; 123: 1093-1106Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We therefore examined the Rb C terminus for potential secondary structure and found a stretch of 21 amino acids with alpha-helix propensity (Rb 895–915; Figures 1G and S3). Deletion of this potential helix (ΔHelix) or disruption of this helix by proline substitution (Q899P or A902P) drastically reduced Rb phosphorylation by cyclin D-Cdk4,6, which was comparable to the phosphorylation of an Rb variant (ΔCdk) lacking all 14 accessible Cdk phosphorylation sites (Figures 1G, 1H, S2A, and S2B). The Rb C-terminal helix is predicted to have one face composed primarily of hydrophobic residues (Figure 1G). We tested whether these residues could form an interface between the Rb C-terminal helix and cyclin D-Cdk4,6 by measuring phosphorylation of alanine substitution mutants by cyclin D-Cdk4. Rb phosphorylation was disrupted when individual predicted interface residues were mutated but was unaffected when individual adjacent residues were mutated (Figure S2C). For example, the impact of arginine-to-alanine substitutions depended on which face of the helix it occurred on (Figures 1G and 1H; compare R908A versus R910A). Combined alanine substitution of three of the core interface residues (F897, L901, and R908), denoted as RbHelix mut., disrupted phosphorylation of Rb by cyclin D-Cdk4,6 similar to that of RbΔHelix (Figures 1G, 1H, and S2D). To test whether the orientation of the helix was important for phosphorylation, we reversed the primary sequence of the helix within the C terminus of full-length Rb. This variant (rev. Helix) was poorly phosphorylated by cyclin D-Cdk4, s" @default.
• W2952456576 created "2019-06-27" @default.
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• W2952456576 date "2019-05-01" @default.
• W2952456576 modified "2023-10-14" @default.
• W2952456576 title "Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein’s C-Terminal Helix" @default.
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• W2952456576 doi "https://doi.org/10.1016/j.molcel.2019.03.020" @default.
• W2952456576 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6800134" @default.
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