The Rb networkJames DeGregoriDepartment of Biochemistry and MolecularGenetics, University of Colorado Health ScienceCenter, Box C229, 4200 East 9th Avenue, Denver,CO 80262, USA

Journal of Cell Science 117, 3411-3413Published by The Company of Biologists 2004doi:10.1242/jcs.01189

A key component of the machinerythat regulates cell cycle entry andprogression in mammalian cells is theretinoblastoma protein (Rb), whichfunctions as a barrier to inappropriatecell cycle progression. The cyclin-dependent kinase (CDK) pathwaycontrolling Rb is deregulated in mosthuman tumors through deregulatedexpression of cyclins, inactivation ofCDK inhibitors such as p16Ink4a or

mutation of Rb itself (Sears and Nevins,2002; Sherr and Roberts, 1999). Theposter highlights some of the pathwaysthat regulate Rb activity, as well asthe mechanisms by which Rbregulates proliferation, apoptosis anddifferentiation.

Rb is a member of a gene familyencoding structurally and functionallysimilar proteins, which include the Rb,p107 and p130 proteins (Stevaux andDyson, 2002). Like Rb, p107 and p130are regulated during the cell cycle byCDK phosphorylation, although thereare clear differences at the levels of bothfunction and expression. In addition, Rbfamily members associate with thecellular transcription factor E2F,negatively regulating E2F-dependenttranscription. E2F activity plays crucialroles in cell cycle progression by

regulating the transcription of genesinvolved in cell cycle regulation, DNAreplication and mitosis (DeGregori,2002; Trimarchi and Lees, 2002). E2Ftranscription factors form variousheterodimers that are each composed ofone E2F subunit and one DP subunit.E2F1, E2F2 and E2F3 family memberspredominantly associate with Rb,whereas E2F4 associates with Rb, p107and p130. E2F5 appears to associatepredominantly with p130. E2F6 andE2F7 appear to repress transcriptionvia Rb-independent mechanisms. Forsimplicity, I use Rb here to refergenerically to all three Rb familymembers.

Starting in late G1 phase of the cellcycle, Rb is heavily phosphorylated untilmitosis. Hypophosphorylated Rb is theactive form of Rb that negatively

Cell Science at a Glance 3411

(See poster insert)

Journal of Cell Science 2004 (117, pp. 3411-3413)

SUV39

PP

Rb

HeterochromatinDifferentiation

Closed chromatin

Open chromatin

Quiescence

Cell cycl eprogress ionor apoptosis

Rbor

Rb

PP P P

PP PP

p21 mRNA

p15 mRNA

Ras

SMADSMEK

E2F1/2/3

TNF-α

TGFβ

DP

AKTAKT

p27

SCF

E2F

SCF

p27

jcs.biologists.org

Rb

CDK4/6Cyclin D

Cyclin D CDK4/6

CDK2

+

Cyclin E

HATAbl*

Abl

Caspases

Cytoplasm

Nucleus

HDAC

CBFA1

Cyclin D1

James DeGregori

Apoptosis

Growth factors

p21

p27

p21

p27p21

Myc

Raf

p16

p15p15

p15

p15

DP

E2F4/5 DP

Rb E2F4/5 DP

ERK

ERKPI3K

GSK3β

RbRb

P

Myc

Cyclin D1

Target gene

Target geneTarget gene

Target gene

RbHP1

Me

Ub

Ub

Cul1

Cyclin D1mRNA

p15/p21

3412

regulates E2F and cell cycle entry. Thehyperphosphorylation of Rb during G1progression is largely carried out byCDKs. Specifically, in mid-G1 phase,Rb is first phosphorylated by Cyclin-D-dependent CDKs (CDK4 or CDK6),which associate with one of three D-typecyclins. The D-type cyclin-CDKs arehighly responsive to growth factor (GF)stimulation at several levels. Theseinclude synthesis of their subunits,association with inhibitory proteins suchas cyclin kinase inhibitors (CKIs),assembly of the subunits, and cyclinstability (Sherr and Roberts, 1999). Boththe transcription of the cyclin D1 geneand assembly of cyclin D1 with CDK4or CDK6 depend on Ras activation.Furthermore, increased Myc activity inearly to mid G1 phase promotes thetranscription of the genes that encodecyclin D1, cyclin D2 and CDK4 (Searsand Nevins, 2002). In a quiescent cell,the kinase GSK3β phosphorylates cyclinD1, resulting in its cytoplasmic retentionand degradation. Following GF receptor(GF-R) signaling via Ras andphosphoinositide 3-kinase (PI 3-kinase),the kinase AKT (also known as PKB)inhibits GSK3β activity, allowing theaccumulation of cyclin D1 in the nucleus(Sherr and Roberts, 1999).

CKIs of the Ink4 family (p16Ink4a,p15Ink4b, p18Ink4c and p19Ink4d)specifically associate with CDK4 andCDK6, blocking the kinase active siteand preventing association with cyclins.By contrast, CKIs of the CIP/KIP family(p21Cip1, p27Kip1 and p57Kip2) associatewith and potently inhibit cyclin-E- andcyclin-A-dependent CDKs. Associationof the cyclin-D–CDK4/6 complex withp21Cip1 and p27Kip1 following GFactivation is required for cyclin-E–CDK2 activation, sequestering theseCKIs away from cyclin-E–CDK2.Interestingly, p21Cip1 and p27Kip1 arealso required for the assembly andnuclear accumulation of cyclin-D–CDKcomplexes (Sherr and Roberts, 1999).The CKIs are controlled at multiplelevels by extra- and intra-cellularmediated signaling. TGFβ signaling,which generally inhibits cell cycleprogression, increases the transcriptionof the p15-encoding Ink4b and p21-encoding Cip1 genes. Similarly, cellularstress or DNA damage can activate p53,which promotes the transcription of

Cip1, leading to CDK inhibition, Rbactivation (reduced phosphorylation)and cell cycle arrest. Furthermore, theRas-MAPK (mitogen activated proteinkinase) pathway may mediate increasedp16Ink4a expression during senescenceinduction. By contrast, AKT activationby GF signaling results in increasedcytoplasmic localization of p21Cip1 andp27Kip1 (Zhou and Hung, 2002). Finally,Myc promotes the transcription of Cul1,which encodes a component of the SCFubiquitin ligase that promotes thedegradation of p27Kip1 protein,contributing to p27Kip1 downregulationduring G1.

The sequential and combinedphosphorylation of Rb by cyclin-D- andcyclin-E-dependent CDKs contributes toinactivation of Rb (Sherr and Roberts,1999; Stevaux and Dyson, 2002).Cyclin-A-dependent CDK activity mightalso contribute to the maintenance ofRb inactivation during S-G2 phaseprogression. The dephosphorylation ofRb is also important to reactivate Rb(although Rb might never be totallyunphosphorylated), either followingmitosis or in response to GF withdrawal,and appears to be mediated by thecombined action of phosphatasestogether with CDK inactivation. Rb isalso regulated following apoptoticstimulation by caspase-mediatedcleavage, which leads to Rb degradation(Chau and Wang, 2003). Indeed, Rbcleavage has been shown to be criticalfor TNFα-induced apoptosis. Rb cannegatively regulate apoptosis byassociating with and inhibiting of theAbl and JNK kinases, as well as byinhibiting the expression of pro-apoptotic E2F target genes such as thoseencoding the p53 family member p73,APAF1 and some caspases (Chau andWang, 2003). Similar roles for p107 andp130 in negatively regulating apoptosishave not been demonstrated.

GF-dependent activation of cyclin–CDK-mediated Rb phosphorylation andE2F activation are necessary (butprobably not sufficient) for cell cycleentry and progression. Association of Rbwith E2F masks the transcriptionalactivation domain of E2F. Importantly,Rb also functions as an activetranscriptional repressor by recruitingvarious cofactors, many of which are

involved in remodeling chromatin(Stevaux and Dyson, 2002). Rb-E2Frecruits histone deacetylases (HDACs)to E2F target promoters; these removeacetyl groups from histone proteins atthe promoter, contributing to a closedchromatin state and transcriptionalrepression. In quiescent cells, E2F-binding sites at E2F-regulated promotersare occupied by E2F4, p130 and HDAC,and this coincides with reduced histoneH3 acetylation and decreased geneexpression. Following GF stimulation,in late G1 phase these same E2F-bindingsites become occupied by E2F1, E2F2and E2F3, and this coincides withincreased histone H3 acetylation andtranscription, which is consistent withthe ability of E2Fs to associate withhistone acetyl transferases (HATs). Rbfamily members also appear to mediategene repression via the recruitment ofPolycomb group (PcG) proteins andcomponents of the Swi/Snf chromatin-remodeling complexes (Stevaux andDyson, 2002).

Notably, in several studies, investigatorshave shown that Rb (as opposed to p107and p130) is not present at E2F-regulatedpromoters in either quiescent or GFactivated cells. By contrast, Rb is foundassociated with these promoters insenescent cells, which results instable repression of E2F-dependenttranscription via the recruitment ofthe SUV39 histone methytransferase(HMT). SUV39 methylation of histoneH3 results in the recruitment ofheterochromatin protein 1 (HP1),which promotes the formationof heterochromatin. SUV39-mediatedmethylation of histone H3 might requireprior deacetylation of the same Lys9residue on histone H3 by HDACactivities. Notice that the role ofHDAC, HMT and chromatin-remodelingcomplex (such as Swi/Snf) activities inRb-mediated repression during the cellcycle, quiescence or senescence have notbeen fully established. The complexesshown should therefore not be interpretedto reflect, for example, distinct roles forHDACs and HMTs in Rb-mediatedgene repression during quiescence andsenescence, respectively.

Rb has also been shown to promote thetranscription of differentiation mediators– for example, following its recruitment

Journal of Cell Science 117 (16)

by the CBFA1 transcription factor topromoters of genes encoding osteogenicfactors and by C/EBP proteins topromoters of genes encoding adipogenicfactors (Thomas et al., 2003). Thus, incontrast to p107 and p130, Rb may playa more specific role in regulating geneexpression during differentiation andsenescence. Finally, Rb and E2Fs havebeen shown to associate near replicationorigins in both mammalian andDrosophila model systems, whichsuggests that they might have directroles in regulating DNA replication(Stevaux and Dyson, 2002).

ReferencesChau, B. N. and Wang, J. Y.(2003). Coordinatedregulation of life and death by RB. Nat. Rev.Cancer3, 130-138.DeGregori, J. (2002). The genetics of the E2Ffamily of transcription factors: shared functionsand unique roles. Biochim. Biophys. Acta1602,131-150.Sears, R. C. and Nevins, J. R.(2002). Signalingnetworks that link cell proliferation and cell fate.J. Biol. Chem.277, 11617-11620.Sherr, C. J. and Roberts, J. M.(1999). CDKinhibitors: positive and negative regulators of G1-phase progression. Genes Dev.13, 1501-1512.Stevaux, O. and Dyson, N. J.(2002). A revisedpicture of the E2F transcriptional network and RBfunction. Curr. Opin. Cell Biol.14, 684-691.Thomas, D. M., Yang, H. S., Alexander, K. and

Hinds, P. W. (2003). Role of the retinoblastomaprotein in differentiation and senescence. CancerBiol. Ther.2, 124-130.Trimarchi, J. M. and Lees, J. A. (2002). Siblingrivalry in the E2F family. Nat. Rev. Mol. Cell. Biol.3, 11-20.Zhou, B. P. and Hung, M. C. (2002). Noveltargets of Akt, p21(Cipl/WAF1), and MDM2.Semin. Oncol.29, 62-70.

Cell Science at a Glance 3413

Cell Science at a Glance on the WebElectronic copies of the poster insert areavailable in the online version of this articleat jcs.biologists.org. The JPEG images canbe downloaded for printing or used asslides.

Commentaries

JCS Commentaries highlight and critically discuss recent exciting work that will interest thoseworking in cell biology, molecular biology, genetics and related disciplines. These short reviewsare commissioned from leading figures in the field and are subject to rigorous peer-review andin-house editorial appraisal. Each issue of the journal usually contains at least two Commentaries.JCS thus provides readers with more than 50 Commentaries over the year, which cover thecomplete spectrum of cell science. The following are just some of the Commentaries appearingin JCS over the coming months.

Holiday junction resolvases Paul Russell

Roles of the centrosome Michel Bornens

Stem cell therapy Helen Blau

IQGAP Kozo Kaibuchi

Dorsal closure Daniel Kiehart

Signal integration Michael Rosen

Kinetochore-microtubule interactions William C. Earnshaw

Electron tomography Wolfgang Baumeister

Myoblast fusion Grace K. Pavlath

Zyxin Mary Beckerle

RNA-directed DNA methylation Judith Bender

Signalling signatures Norbert Perrimon

Necrotic-like cell death Monica Driscoll

Although we discourage submission of unsolicited Commentaries to the journal, ideas for futurearticles – in the form of a short proposal and some key references – are welcome and should besent to the Executive Editor at the address below.

Journal of Cell Science, Bidder Building, 140 Cowley Rd, Cambridge, UK CB4 0DLE-mail: jcs@biologists.com; http://jcs.biologists.org