(1998) 17, 3365 ± 3383 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Role of the family, pRB, p107 and p130 in the negative control of

Xavier GranÄ a*,1, Judit Garriga1 and Xavier Mayol2

1Fels Institute for Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, 3307 North Broad St., Philadelphia, PA19140, USA; 2Unitat de Biologia Cel.lular i Molecular, Institut Municipal d' Investigacio Medica (IMIM), Dr. Aiguader 80, 08003 Barcelona, Spain

The retinoblastoma family of , also known as capable of inhibiting growth in certain cell types, and pocket proteins, includes the product of the retinoblas- this inhibition depends on their ability to associate with toma susceptibility gene and the functionally and a number of cellular proteins. In general terms, many structurally related proteins p107 and p130. Pocket of the targets of pocket proteins are either proteins control growth processes in many cell types, and factors or proteins involved in the regulation of this has been linked to the ability of pocket proteins to transcription (reviewed in White, 1997; Johnson and interact with a multitude of cellular proteins that Schneider-Broussard, 1998; Mayol and GranÄ a, 1998; regulate at various levels. By regulating Yee et al., 1998). For instance, pRB has been shown to gene expression, pocket proteins control inhibit RNA transcription by the three cellular RNA progression, cell cycle entry and exit, cell di€erentiation polymerases. One prominent function shared by the and . This review will focus on the mechanisms three pocket proteins is the negative regulation of a of regulation of pocket proteins and how modulation of family of transcription factors collectively designated as pocket protein levels and status regulate (Johnson and Schneider-Broussard, 1998). E2F association with their cellular targets. The coordinated family members regulate the transcription of several regulation of pocket proteins provides the cells with a genes, whose products are required either for the competence mechanism for passage through certain cell regulation of G1 and S phases of the cell cycle or for growth and di€erentiation transitions. DNA metabolism. Thus, by negatively regulating E2F, pocket proteins seem to control, at least in part, the Keywords: CDK; CKI; transcriptional regulation; progression through the cell cycle and the cell cycle ; cell cycle entry/exit transitions. Moreover, pocket proteins have also been implicated in the process of cell differentia- tion in various cell types.

Introduction Di€erential phosphorylation of pocket proteins Inactivation of both copies of the retinoblastoma gene is associated with the development of The three members of the pRB family are phospho- in humans. In addition, other types of tumors also proteins and their phosphorylation status is modulated exhibit functional inactivation of the retinoblastoma during the cell cycle as well as at the cell cycle entry gene product, pRB, together with defects in other and exit transitions in mammalian cells (Buchkovich et genes. Indeed, deregulation of the pRB pathway is very al., 1989; Chen et al., 1989; DeCaprio et al., 1989; common in human cancer (for review see Weinberg, Mihara et al., 1989; Beijersbergen et al., 1995; Mayol et 1995; Mulligan and Jacks, 1998). These data together al., 1995; Xiao et al., 1996). Multiple sites are with the ability of pRB to restrict growth when phosphorylated in pRB and most likely in p107 and overexpressed in certain cell types indicate that it p130. All sites identi®ed thus far fall within the functions as tumor suppressor, at least in part, by consensus site of phosphorylation by -Depen- negatively regulating cell division. Identi®cation of two Kinases (CDKs), which are primarily serine/threonine, members of the retinoblastoma family of proteins, proline-directed sites. Most of the work done to date p107 (Ewen et al., 1991; Zhu et al., 1993) and p130 has focussed on pRB and most of its in vivo (Hannon et al., 1993; Li et al., 1993; Mayol et al., phosphorylation sites are known. However, less is 1993), which share structural and functional similarity known about which kinases phosphorylate each one of with pRB, led to numerous studies seeking to these sites at a particular time, although a large body understand whether these two proteins where also of evidence indicates that cell cycle-dependent phos- tumor suppressors. However, mutations on these genes phorylation of pocket proteins is catalyzed by a subset are rare, and their role as tumor suppressor has not of cyclin/CDK holoenzymes that are sequentially been demonstrated. As pRB, both p107 and p130 are activated during the cell cycle (reviewed in Bartek et al., 1996; Mayol and GranÄ a, 1997, 1998). Phosphoryla- tion of pocket proteins in mid-to-late is functionally important because it is associated with the *Correspondence: X GranÄ a, Fels Institute for Cancer Research and loss of the growth suppressive activities of the three Molecular Biology, Temple University School of Medicine, AHP bldg., room 553, 3307 North Broad St., Philadelphia, PA 19140, pocket proteins. The growth suppressive activity of USA pocket proteins is mediated through the association The pRB family in negative cell growth control XGranÄa et al 3366 with a number of cellular proteins, among them, the that phosphorylation of Ser-795 by /CDK-4 members of the E2F family of transcription factors, complexes is necessary for inactivation of the growth which regulate the expression of a number of genes suppressor activity of pRB in SAOS-2 cells (Connell- required for cell cycle progression. Crowley et al., 1997). These results highlight the importance of the phosphorylation of particular sites versus the idea that extensive phosphorylation of pRB Cyclin/CDK complexes collaborate to phosphorylate is required for its inactivation, and suggest that cyclin pocket proteins D-associated kinase activities are essential for the Progression through mid G1 and S phases from a functional inactivation of pRB. quiescent stage or from a previous mitotic phase is A di€erential role for cyclin D1 and in pRB associated with the sequential activation of a subset of phosphorylation was suggested by experiments con- cyclin-dependent kinases (reviewed in GranÄ aand ditionally expressing these cyclins in serum starved and Reddy, 1995). D-type cyclin/CDK complexes are re-stimulated Rat-1 cells. Although premature ectopic activated upon stimulation of quiescent expression of both cyclins reduced the length of the G1 cells and are kept active through G1. Cyclin E/CDK2 phase, only cyclin D1 expression led to premature kinase activity increases from mid-to-late G1. Activa- hyperphosphorylation of pRB (Resnitzky et al., 1994; tion of these two subtypes of cyclin/CDK complexes Resnitzky and Reed, 1995). A di€erent experimental correlates with hyperphosphorylation of the three approach was based on selectively blocking the activity pocket proteins from mid-to-late G1. /CDK2 of D type cyclin/CDK and cyclin E/CDK2 complexes complexes, whose activities peak in , are also using the CDK inhibitor and a dominant negative likely to contribute to the maintenance of the CDK2 mutant (dnCDK2) respectively (Lundberg and phosphorylation pool of pocket proteins. This hyper- Weinberg, 1998). These experiments suggested that phosphorylated state is kept through the remaining of endogenous D-type cyclins phosphorylate pRB only the cell cycle to mitosis, where these forms shift to a partially and that cyclin E/CDK2 complexes cannot hypophosphorylated state by the action, at least in the phosphorylate pRB without previous phosphorylation case of pRB, of a protein phosphatase (reviewed by by D-type cyclin/CDK complexes. Interestingly, Ludlow and Nelson, 1995). All these cyclin/CDK phosphorylation of pRB in vivo by presumably complexes are capable of phosphorylating each pocket endogenous D-type cyclin/CDK complexes in the protein in vitro. In the case of pRB (this has not been presence of dnCDK2, which block cyclin E/CDK2 addressed for p107 and p130), phosphorylation in vitro activity, is not sucient to abrogate binding to GST- occurs at sites known to be phosphorylated in vivo E2F in vitro. This is in agreement with the ability of (Lees et al., 1991; Lin et al., 1991; Kato et al., 1993; dnCDK2 to repress E2F-dependent transactivation Kitagawa et al., 1996; Knudsen and Wang, 1996; (Lundberg and Weinberg, 1998). Connell-Crowley et al., 1997; Zarkowska and Mitt- Phosphorylation of speci®c sites on pRB has also nacht, 1997; Zarkowska et al., 1997), and abrogates been linked to the di€erential abrogation of the pRB interaction with E2F complexes (Suzuki-Takaha- interaction between pRB and a set of pRB associated shi et al., 1995; Zhu et al., 1995b). Similarly, isolated proteins. Phosphorylation of serines 807 and 811 is native p130/E2F-4 complexes are also disrupted by required to abrogate the interaction between pRB and cyclin/CDK complexes in vitro (Mayol et al., 1996). In c-Abl, while phosphorylation of threonines 821 and agreement with the in vitro experiments, ectopic 826 is required to abolish pRB binding to proteins overexpression of cyclins D1, E or A together with containing LXCXE motifs such as the SV40 large T pRB lead to hyperphosphorylation of pRB and antigen (Knudsen and Wang, 1996). In this regard, abrogates pRB growth inhibitory activity in SAOS-2 threonines 821 and 826 are phosphorylated in vitro by cells, which do not express a functional pRB (Hinds et cyclin A/CDK2 and cyclin D1/CDK4 respectively. al., 1992; Horton et al., 1995). It is likely, however, Phosphorylation by either complex will preclude pRB that in vivo abrogation of pocket protein/E2F binding to T antigen, however only cyclin A/CDK2 complexes requires the sequential action of D-type complexes can disrupt preformed complexes. This cyclins and cyclin E (Lundberg and Weinberg, 1998). seems to be due to the inability of cyclin D1/CDK4 Recent studies have revealed di€erences in the to phosphorylate threonine 826 if pRB is pre-bound to phosphorylation of speci®c sites on pRB by the above a LXCXE containing protein (Zarkowska and mentioned G1/S cyclin dependent kinases (Kitagawa et Mittnacht, 1997). al., 1996; Connell-Crowley et al., 1997; Zarkowska and The apparent contradictory conclusions in some of Mittnacht, 1997; Zarkowska et al., 1997). However, the above mentioned studies are certainly due to a these results are controversial since there are reports that diculty of studying phosphorylation of pRB in its do not seem to agree in the phosphorylation speci®city of physiological cellular context. However, a tentative certain sites by a particular cyclin/CDK complex. model can be drawn (Figure 1). It seems likely that A particular residue in pRB, Ser-795, which is each G1/S cyclin/CDK complex phosphorylates pRB eciently phosphorylated by CDK4/cyclin D1 com- and related family members at particular sets of plexes, is not phosphorylated by either CDK2/ or phosphorylation sites and at speci®c times during CDK3/cyclin complexes in vitro (Connell-Crowley et progression through the cell cycle. The nature of the al., 1997). CDK2 or CDK3 phosphorylate multiple proteins associated to a particular pocket protein, or other sites resulting in hyperphosphorylated pRB, and the absence of these proteins is very likely to a€ect the this action results in the abrogation of the pRB/E2F ability of cyclin/CDK complexes to phosphorylate interaction in vitro. However, this in vitro hyperpho- particular sites. In addition, previous phosphorylation sphorylated pRB form is still capable of suppressing by a cyclin/CDK complex, might result in conforma- growth when microinjected in SAOS-2 cells, suggesting tional changes on the pocket protein that convert it The pRB family in negative cell growth control XGranÄa et al 3367

Figure 1 Di€erential regulation of pocket protein phosphorylation and protein levels in quiescent cells re-entering the cell cycle. This is a tentative, simpli®ed model that schematizes the modulation of the phosphorylation status and protein levels of pocket proteins (see text for details). (a) In quiescent cells and in cells at the G0/G1 transition p130 is detected primarily as phosphorylated form 2 (lower levels of form 1 are also detected in many cell types), pRB is found hypophosphorylated, whereas p107 levels are low to non-detectable (represented by a crossed molecule). The three pocket proteins are phosphorylated by cyclin /CDKs from mid G1 to the remaining of the cell cycle, most likely by the cooperative action of G1 and S phase cyclin CDK complexes (phosphorylation is represented by black-boxed P(s), to distinguish from G0 p130 forms, plain P(s)). pRB and p107 hyperphosphorylated forms are their predominant state through the rest of the cell cycle (note that hypophosphorylated forms are also detected). p130 phosphorylation to form 3 is followed by dramatic downregulation of p130 levels (represented by a crossed molecule). Concomitantly, p107 expression is upregulated. (b) The periods of activation/inactivation of cyclin/CDK complexes are depicted. (c) Relative changes in the protein levels of each pocket protein. p130 protein levels are dramatically downregulated upon phosphorylation to form 3. p107 protein accumulates rapidly from mid G1 to mitosis. Finally, pRB protein levels remain relatively constant through the cell cycle when compared to either p107 or p130. Indeed, pRB levels increase slightly in certain cell types while decrease in others into a suitable substrate for another CDK. Indeed, as substrate for these CDKs. This conclusion is based on we will discuss later, cyclin/CDK complexes form experiments that demonstrate that the essential function stable complexes with pocket proteins. D-type cyclins of D-type cyclins in G1 depends upon the expression of interact with pocket proteins through their LXCXE a functional pRB. In these experiments, microinjected motif (Dowdy et al., 1993; Ewen et al., 1993; Kato et cyclin D1 anti-sense cDNAs or anti-cyclin D1 anti- al., 1993), while cyclin E and cyclin A/CDK2 bodies were shown to block G1 progression only in cells complexes form stable complexes with p107 and containing functional pRB (Lukas et al., 1994, 1995a; p130, but do not with pRB. Thus, it is conceivable Tam et al., 1994). In agreement with this notion, the that the activity of a particular cyclin/CDK is CKI p16 only blocks G1 progression in cells with necessary to promote and/or negate the action of a functional pRB (Lukas et al., 1995b; Medema et al., di€erent cyclin/CDK complex. Phosphorylation of 1995). Hatekayama and collaborators however, have particular sites, or sets of sites, is likely to result in recently shown that wild type pRB and a phosphoryla- the sequential disruption of pocket protein complexes tion resistant pRB mutant do not block cell cycle containing di€erent transcription factors leading to the progression in at least two di€erent mouse cytokine- orchestrated succession of events required to promote dependent hematopoietic cell lines (a pro-B cell line and cell growth and progression through the cell cycle. myeloid 32Dcl3 cells) when ectopically expressed to supra-physiological levels (Hoshikawa et al., 1998). Instead, p130 eciently blocked cell cycle progression Is pRB the only essential substrate of D-type cyclin/ through its E2F-binding domain in the mouse pro-B cell CDK complexes? line. In these cells, p130 associates with E2F-4 and Although all three known pocket proteins are likely to ectopic overexpression of E2F-4 was sucient to render be phosphorylated by D-type cyclin/CDK complexes, it the cells cytokine-independent (Hoshikawa et al., 1998). has been suggested that pRB is the only essential It would be interesting to learn whether p130 is an The pRB family in negative cell growth control XGranÄa et al 3368 essential target for D-type cyclin/CDK holoenzymes in results in the primary accumulation of p130 form 1 and these cells. Consistent with this, p130 and p107 depletion of p130 form 3, correlating with a TGF-b- containing complexes, but not pRB complexes, are the mediated inactivation of G1 cyclin/CDK complexes. major components of E2F-DNA-binding activities in This transient block in G1 should not be confused with mouse T lymphocytes progressing through the cell cycle a G0 quiescent stage, in which p130 from 2 is the (Cobrinik et al., 1993). In normal mouse T lymphocytes, primary form of p130 in these cells (Mayol et al., however, ablation of p130 or p130/p107 functions, is 1996). In agreement with these results, p130 shift to compensated by p107 or pRB, respectively (Mulligan et form 3 is blocked in serum starved and re-stimulated al., 1998). These experiments suggest that although pRB HaCaT cells progressing through early G1 in the is a critical cell cycle regulator in some cell types, p130 presence of TGF-b. However, p130 form 2 does not function is also critical in certain cell types such as accumulate as a consequence, suggesting that p130 hematopoietic cells. form 2 is not just an intermediate stage of phosphor- ylation due to lack of activation of G1 cyclin/CDK complexes (Mayol et al., 1996). In agreement with the Di€erential regulation of pocket proteins by changes in p130 phosphorylation status described phosphorylation above, cell cycle exit to G0 by serum starvation of p107 and p130 exhibit a simpler pattern of di€erently HaCaT cells results in the accumulation of p130/E2F-4 phosphorylated forms than pRB when resolved by complexes, while a TGF-b-mediated G1 arrest does not SDS ± PAGE followed by Western blot analysis (Belbrahem et al., 1996). (Figure 1). Several pRB forms with di€erent mobility are detected during a complete cell cycle, and the patterns of pRB forms change signi®cantly from cell Modulation of pocket protein levels type to cell type. In contrast, two di€erent p107 forms are detected in most cell types and these forms are Although the levels of pRB are quite constant during generally designated as hypo- and hyperphosphory- the cell cycle and in quiescent cells, levels of p130 and lated p107. In the case of p130, four forms are p107 change dramatically depending upon the stage of detected in most cell types, which are designated the cell cycle (Beijersbergen et al., 1995; Mayol et al., unphosphorylated p130 and phosphorylated p130 1995, 1996). p130 is detected in quiescent cells as two forms 1, 2 and 3 (Mayol et al., 1995). Interestingly, abundant phosphorylated forms (forms 1 and 2). each one of these forms can be associated with a When cells progress through the G0/G1 transition and particular stage of the cell cycle or cell quiescence through mid G1, p130 is hyperphosphorylated to form (Mayol et al., 1996; Garriga et al., 1998). Particular 3, whose levels are dramatically downregulated as cells attention needs to be taken to the antibodies used to move to S phase (Mayol et al., 1995, 1996). In detect phosphorylated forms of pocket proteins in contrast, when cycling cells are forced to exit the cell immunoprecipitation and Western blot experiments. It cycle, p130 protein levels raise sharply (p130 forms 1 is well known that some of the available antibodies and 2) coinciding with the accumulation of p130/E2F (especially some monoclonal antibodies), often recog- complexes (Kiess et al., 1995; Mayol et al., 1996; nize only a subset of forms. Smith et al., 1996; Garriga et al., 1998). The increase As we have discussed above, phosphorylation of the of p130 levels and p130/E2F protein complexes in three pocket proteins during the cell cycle is likely to response to cell cycle exit signals occurs from a period occur through shared cyclin/CDK-dependent path- during the G1 phase previous to the , ways. Activation of cyclin/CDK complexes in mid-to- where otherwise p130 would be phosphorylated to late G1 results in the hyper-phosphorylation of pRB form 3 (Mayol et al., 1996). This was proved in and p107 and in the phosphorylation of p130 to form experiments where synchronous populations of cells at 3. In contrast, at the G1/G0 transition, when cells exit particular stages of the cell cycle were forced to exit the cell cycle to a resting state, p130 phosphorylation the cell cycle. Placing post-restriction point cells, status is modulated di€erently from pRB and p107 which have not reached mitosis, in environmental (Mayol et al., 1996; Garriga et al., 1998). While pRB conditions leading to cell cycle exit did not result in and p107 become hypophosphorylated, p130 is accumulation of p130 or in the accumulation of p130/ detected as phosphorylated forms 1 and 2. This E2F complexes until the post-mitotic G1 phase. These unique modulation of p130 phosphorylation status experiments linked p130 accumulation to the G1/G0 correlates with a dramatic accumulation of p130 transition and not to any transient cell cycle arrest at protein and p130/E2F complexes, which are character- other cell cycle points (Mayol et al., 1996). Since the istic of a quiescent stage. Because this phosphorylation decision to exit the cell cycle does not occur until the event that speci®cally targets p130 is maintained in post-mitotic early G1 phase, accumulation of p130 quiescent cells and readily detected in adult tissues, might regulate the decision to exit the cell cycle. when no G1, S and G2/M cyclin/CDK activities are Accumulation of hypophosphorylated pRB in these detectable, we postulated that a di€erent kinase activity conditions is likely to play a role in the shutting-o€ of targets p130 at the cell cycle exit and during the E2F-dependent transcription (Ikeda et al., 1996). quiescent stage (Mayol et al., 1995, 1996; Garriga et Whether pRB and p130 act individually or a al., 1998). It is still unclear whether this kinase is a concerted action of both proteins regulates this step di€erently regulated CDK family member, or con- is not yet clear, and it might be cell type-dependent versely, another unrelated kinase (reviewed in Mayol (Hoshikawa et al., 1998). The study of mice harboring and GranÄ a, 1998). mutations in genes encoding pocket proteins has A transient block in G1 is induced by TGF-b in provided some clues. However, the compensatory exponentially growing HaCaT cells. This block in G1 ability of the remaining pocket protein(s) to partially The pRB family in negative cell growth control XGranÄa et al 3369 substitute for the absent one has made these studies a number of cell cycle regulators, including p27, cyclin dicult to interpret. D1 and cyclin E, are targeted by the proteasome The levels of p107 are modulated in an opposing machinery upon phosphorylation of particular CDK manner to p130. Very little-to-non p107 is detected in consensus sites (Clurman et al., 1996; Won and Reed, quiescent cells. However, p107 accumulates sharply as 1996; Diehl et al., 1997; Shea€ et al., 1997), it is cells progress through the cell cycle (Beijersbergen et possible that p130 downregulation is mediated by a al., 1995). The increase in p107 protein levels correlate cell cycle stage-dependent proteasome degradation with an increase in p107 mRNA transcripts, indicating pathway. In support of increased degradation of that p107 protein expression is regulated at the p130 during S phase, hyperphosphorylated p130 form transcriptional level (Hurford et al., 1997). Interest- 3 is metabolically labeled more eciently than G0- ingly, p107 is under negative control mediated through phase p130 forms, thus suggesting that the rates of E2F sites on its , suggesting that either pRB, synthesis and phosphorylation are higher in S phase p130 or both are involved in the control of p107 and, therefore, G0-phase p130 forms must be more expression (Zhu et al., 1995c). The existence of stable than p130 form 3 (Kiess et al., 1995; Mayol et connecting regulatory loops among pocket proteins al., 1995). Moreover, preliminary experiments using ensures that their levels oscillate coordinately during proteasome inhibitors block p130 depletion in S the cell cycle and at the G0/G1 and G1/G0 transitions phase, but cell cycle progression through S phase in in a way that allows an orchestrated regulation of the analysed cells was also delayed by these inhibitors common target factors (reviewed in Mayol and GranÄ a, and the involvement of the proteasome complex may 1998). be indirect (Smith et al., 1998). In this context, we On the other hand, very little oscillation of the have found that cellular treatments that delay the levels of p130 mRNA transcripts is observed in progression of the cell cycle through S phase, even a quiescent and cycling cells (Richon et al., 1997; slight e€ect, also delay the downregulation of p130 Smith et al., 1998), indicating that changes in protein (unpublished observations). Thus, although the above levels are primarily post-transcriptional. Thus, p130 cited hypothesis is an attractive possibility, the levels are regulated by the rate of protein synthesis mechanism of p130 downregulation is yet to be and/or by changes in protein stability linked to clearly demonstrated. Finally, at the cell cycle exit, speci®c cell cycle stages. In this regard, there is a dramatic accumulation of p130 occurs without striking correlation between the phosphorylation changes in p130 mRNA levels, suggesting that p130 status of p130 and its protein levels, suggesting that accumulation is regulated at a post-transcriptional phosphorylation of speci®c sites regulates the stability level. Consistent with this, cell cycle exit phosphoryla- of the p130 protein. The phosphorylation events tion of p130 indeed results in stabilization of the p130 leading to the generation of p130 form 3 in mid G1 protein since p130 form 2 accumulates while the are followed by dramatic downregulation of p130 metabolic labeling rates of p130 are reduced during (Mayol et al., 1995, 1996), but the mechanisms quiescence (Mayol et al., 1995, 1996; Garriga et al., mediating this downregulation are still unclear. Since 1998).

Table 1 Cellular protein targets of the pRB family of proteins (see text for references) Associated protein Biochemical function Pocket protein Biological role CDK subunit pRB Cell cycle Cyclins A and E CDK subunits p107, p130 Cell cycle RbK Protein kinase pRB Cell cycle? E2F family Transcription factors pRB, p107, p130 Cell cycle c-Jun pRB Cell cycle? ATF-2 Transcription factor pRB TGF-beta expression c-, N-Myc Transcription factors pRB, p107 Cell cycle? Spl Transcription factor pRB, p107 Cell cycle? Abl Nuclear tyrosine kinase pRB Cell cycle? Nuclear protein pRB pRB inhibitor? MCM7 DNA replication licensing pRB, p107, p130 DNA replication inhibition? HNuc Nuclear protein pRB Mitosis? RBAp48 Transcription factor- pRB Growth inhibition? AhR Transcription factor pRB TCDD-growth arrest TAFII250/TFIID Transcription factor pRB Transcription TFIIIB RNA pol III-Transcription factor pRB tRNA synthesis UBF RNA pol I-Transcription factor pRB rRNA synthesis HDAC1 deacetylase pRB, p107, p130 Transcription MyoD Transcription factor pRB, p107 Muscle di€erentiation HBP1 Transcription factor pRB, p130 Muscle di€erentiation p202 Transcription factor pRB Muscle di€erentiation C/EBP, NF-IL6 Transcription factor pRB Adipocyte di€erentiation NRP/B Nuclear matrix pRB Neuronal di€erentiation PLH proteins Transcription factor pRB, p107, p130 Homeosis? BRG1 Transcription factor pRB ? hBRM Transcription factor- pRB Glucocorticoid response? Id-2 Transcription factor-corepressor pRB Cell cycle? AP-2 Transcription factor pRB Epithelial di€erentiation? Trip230 THR-coactivator pRB Thyroid hormone response The pRB family in negative cell growth control XGranÄa et al 3370 Biochemical consequences of the regulation of pocket composed of CDK2, which is most active in late S proteins and G2 phases (Mayol et al., 1995; Vairo et al., 1995). Similarly, p107 associates with cyclin E/CDK2 and As discussed later, the biochemical action of pocket cyclin A/CDK2 complexes in late G1 and during the S proteins is largely dependent on their ability to phase (Lees et al., 1992; Shirodkar et al., 1992; associate and thereby modulate the activities of Chittenden et al., 1993; Cobrinik et al., 1993; Schwarz cellular proteins (Table 1). These associations involve et al., 1993). These data are in support of an active role a wide range of transcription factors, as well as of pocket proteins during stages when they are thought enzymes such as , through which to be inactivated by CDK-mediated hyperphosphoryla- pocket proteins are thought to regulate gene expres- tion. As discussed earlier, multiple amino acid sites in sion. Indeed, through interactions with various pocket proteins are coordinately phosphorylated by transcription factors, pRB family members are capable diverse CDKs, and suppression of binding abilities to of regulating the activity of the three RNA poly- particular cellular proteins may thus depend on the merases (reviewed by Dynlacht, 1995; White, 1997). status of key phosphorylation sites. Moreover, Pocket proteins also associate with tissue speci®c although direct evidence for the in vivo binding of transcription factors including MyoD, HBP1, C/EBP, hyperphosphorylated pocket protein forms to tran- and NRP/B, among others, which may be important scription factors or to other cellular proteins has not mediators of the biological role of pocket proteins in been reported, hyperphosphorylation of p107 and p130 cell di€erentiation processes. Moreover, pocket pro- does not a€ect their binding ability to the SV40 large T teins associate with di€erent cyclin/CDK complexes, antigen-a LXCXE-containing viral protein. In contrast, perhaps, modulating their activity, and recent ®ndings binding of pRB to this viral protein is largely suggest that they may also interact directly with dependent on the phosphorylation of threonine 821 components of the DNA replication machinery. Thus, not conserved in p130 or p107 (Knudsen and Wang, pocket proteins appear to act at diverse cellular levels 1998). These observations indicate that cyclin-CDK to control cell growth and di€erentiation processes. activation in G1 may not result in the absolute disruption of pocket protein associations and that hyperphosphorylated pocket protein forms still retain Association with cellular proteins the ability to form certain protein complexes. The association of pocket proteins with their cellular Pocket protein association with cellular proteins is partners is dependent, in many cases, on the also regulated by modulation of the pocket protein phosphorylation status of the pocket protein. Only levels in the cell. This is particularly important in the unphosphorylated or hypophosphorylated pocket case of p130 and p107, which display a high degree of protein forms are able to associate with most of their biochemical similarity and show opposite patterns of targets. The inference that lack of cyclin-CDK expression in diverse instances of cell growth and phosphorylation on pocket proteins is determinant in di€erentiation (reviewed in Mayol and GranÄ a, 1997, allowing such protein associations is based on results 1998). of experiments in which hyperphosphorylation of Finally, availability of cellular targets to pocket pocket proteins correlated with protein complex proteins is a determinant in the modulation of cellular disruption. Conversely, pocket protein hypophosphor- pathways that depend on pocket protein function. ylation correlates with the appearance of such Thus, in processes such as cell di€erentiation that complexes. In support of this, in vitro experiments require pocket protein-mediated regulation of gene have shown that CDK activities directly disrupt or expression, tissue-speci®c transcription factors need to impede pocket protein/E2F complexes (Suzuki-Taka- be expressed and exposed to pocket proteins in order hashi et al., 1995; Zhu et al., 1995b; Mayol et al., to be biochemically activated or inactivated. 1996). The kinetics of activation/inactivation in vivo of the cyclin/CDK complexes used in these in vitro Transcriptional repression mediated by E2F experiments correlates with the status of pocket protein phosphorylation observed during cell cycle The E2F family of transcription factors is composed of progression and growth arrest. Although direct proof six members (E2F-1 to -6) that form DNA-binding for this phosphorylation-dependent association with heterodimers with members of the related DP family of cellular proteins has not been assessed in all the co-activators (DP-1 to DP-3). In proliferating cells, instances, this mechanism is assumed to be a general E2F proteins activate transcription of several genes mechanism governing interactions between these important for cell cycle progression (for recent reviews proteins (reviewed in Mayol and GranÄ a, 1997, 1998; see Johnson and Schneider-Broussard, 1998; Mayol Johnson and Schneider-Broussard, 1998; Yee et al., and GranÄ a, 1998, and references therein). In contrast, 1998). in growth-inhibited cells as well as at initial stages of In some cases, association of pocket proteins with G1 progression, E2F proteins are targeted by pocket certain cellular proteins may apparently be without the proteins and are converted into transcriptional restrain imposed by CDK phosphorylation in G1. repressors of many genes that are positively regulated These cases include the association with certain cyclin/ during the cell cycle. The recently identi®ed E2F-6, CDK complexes by direct binding, which occurs during di€ers from other members of the family in that it does cell cycle phases when the activities of these kinases are not posses a pocket protein binding domain, yet, seems high and pocket proteins are primarily hyperpho- to act as a repressor of E2F-site-dependent transcrip- sphorylated (Figure 1). In particular, hyperphosphory- tion (Morkel et al., 1997; Cartwright et al., 1998; lated p130 possesses a transient histone H1-associated Gaubatz et al., 1998; Trimarchi et al., 1998). Pocket kinase activity during the cell cycle, most likely proteins associate di€erentially with certain E2F family The pRB family in negative cell growth control XGranÄa et al 3371 members both in vivo and in vitro, p130 and p107 1998). The role of these E2F-regulated genes in cell exclusively targeting E2F-4 and E2F-5, and pRB growth processes, together with the fact that activation targeting E2F-1, E2F-2, E2F-3 and E2F-4 (Figure 2; of E2F family members is sucient to induce DNA reviewed in Johnson and Schneider-Broussard, 1998; replication and that pocket protein suppressive Mayol and GranÄ a, 1998). The genes transcriptionally function on the cell cycle is largely dependent on the regulated by E2F transcription factors include genes interaction with E2F proteins (reviewed in Johnson coding for cell cycle regulatory proteins: cyclin E and Schneider-Broussard, 1998), indicates that pocket (Ohtani et al., 1995; Botz et al., 1996; Geng et al., proteins and E2F play a central role in the control of 1996), cyclin A (Schulze et al., 1995; Zerfass et al., the cell division cycle. Pocket protein phosphorylation 1995; Huet et al., 1996; Philips et al., 1998), CDC2 by cyclin/CDKs during the G1/S transition disrupts (Dalton, 1992; Tommasi and Pfeifer, 1995), CDC25C E2F-pocket protein complexes, both, relieving their (Zwicker et al., 1995a, b), the CDK inhibitor repressive action on gene expression and releasing (Hiyama et al., 1998) and protooncogenes such as Myc active E2F transcription factors. Consequently, activa- and Myb (reviewed in Slansky and Farnham, 1996). tion of E2F in G1 allows the transcription of genes Interestingly, p107 (Zhu et al., 1995c), E2F-1 (Hsiao et required for cell cycle progression. In this scenario, a al., 1994; Johnson et al., 1994; Neuman et al., 1994; number of a€erent growth signals, either activating or Johnson, 1995; Smith et al., 1996), and E2F-2 genes suppressing growth, are integrated by cyclin-CDKs (Smith et al., 1996; Sears et al., 1997) are also under leading to modulation of the phosphorylation status of E2F transcriptional control during the cell cycle, which pocket proteins, which ultimately determines the may have relevant implications in the coordination of relative action of E2F as a transcriptional activator individual pocket protein functions and is discussed or repressor. Therefore, it is widely accepted that this later in this section. Moreover, proteins involved in pathway constitutes one of the main steps in the DNA metabolism and some constituents of DNA decision of passage through the G1 restriction (R) replication complexes are also transcriptionally regu- point, from which cells are committed to cell cycle lated by E2F proteins: dihydrofolate reductase progression independently of external growth signals. (DHFR) (Means et al., 1992; Slansky et al., 1993), Conversely, if growth inhibiting signals dominate, this thymidine kinase (TK) (Dou et al., 1992; Ogris et al., pathway is blocked at particular points and, eventually, 1993), thymidylate synthase (TS) (Johnson, 1992), cells may chose to exit the cell cycle. histone H2A (Oswald et al., 1996), Orc1 (Ohtani et Some of the genes whose products are required for al., 1996), and CDC6 (Ohtani et al., 1998; Yan et al., cell proliferation are known to be transcriptionally

Figure 2 Regulation of the expression of genes containing E2F sites by E2F and E2F/pocket protein complexes. (a) Transcriptional repression mediated by E2F. E2F family members heterodimerize with any DP protein. pRB associates with E2F-1 to 4/DP heterodimers. p130 and p107 associate with both E2F-4/DP and E2F-5/DP heterodimers. E2F-6 does not contain a pocket protein binding domain. E2F/DP/pocket protein complexes repress E2F-responsive promoters in G0 and early G1. Repression is released in mid-to-late G1 by cyclin/CDK mediated disruption of pocket protein/E2F interactions. (b) Transcriptional activation mediated by E2F. E2F-1 to 3/DP, and perhaps E2F-4 and 5/DP heterodimers, mediate positive transactivation of E2F responsive genes by binding to E2F-responsive promoters in mid-to late G1 and S phases. These promoters are silent in G0 due to the absence of free E2F complexes The pRB family in negative cell growth control XGranÄa et al 3372 repressed by E2F in non-dividing cells. Since E2F-4 is Johnson, 1995; Smith et al., 1996; Sears et al., 1997). It the main E2F family member found in quiescent cells, is thus assumed that one of the consequences upon and is mostly found in association with p130 in a wide disruption of p130/E2F-4 complexes by G1 cyclin/ range of quiescence and di€erentiation models CDKs is the transcriptional activation of E2F-1 and (reviewed in Johnson and Schneider-Broussard, 1998; E2F-2. Since E2F-1 and E2F-2 speci®cally associate Mayol and GranÄ a, 1998), it is assumed that E2F- with pRB, but not with p130 or p107, and since pRB mediated repression of gene expression, upon cell cycle rarely associates with E2F-4 in the presence of p130/ exit, is primarily exerted by p130/E2F-4 complexes. It E2F-4 complexes, these data again suggest that the must be noted that, pRB has also been reported to pRB-E2F interaction during G1 is largely dependent complex with E2F in some quiescent cells (Corbeil et on previous disruption of p130/E2F-4 complexes. This al., 1995; Halevy et al., 1995; Ikeda et al., 1996; scenario further implicates pRB as a downstream Rampalli et al., 1998). Furthermore, serum-deprived e€ector of p130 function during the G0/G1 transi- pRB7/7 mouse ®broblasts show deregulated expression tion. In contrast, only E2F-3 DNA-binding activities of some E2F-dependent genes, which suggests that re-accumulate in post-mitotic G1 Ref 52 cells (Leone et pRB plays a role similar to p130, at least in the al., 1998). Most importantly, antibodies raised to E2F- repression of certain genes. One of the mechanisms 3, but not anti-E2F-1 antibodies, microinjected in Ref that may be critical in the formation of these major 52 cells synchronized in G2 resulted in inhibition of S p130/E2F-4 complexes is the accumulation of p130 phase-entry of the next cell cycle. Therefore, the protein upon cell cycle exit, probably by protein mechanisms that regulate the expression of E2F- stabilization, and downregulation of p107, whose dependent genes are di€erent in cells entering the cell major partner is E2F-4 (Kiess et al., 1995; Mayol et cycle from a quiescent stage or in proliferating cells. al., 1996; Smith et al., 1996, 1998; Garriga et al., 1998; The p107 gene is also silent in resting cells at least reviewed in Mayol and GranÄ a, 1998). The reason why partially due to E2F-dependent transcriptional repres- pRB is not found appreciably associated with E2F sion, and is re-activated in mid G1 during cell cycle after cell cycle exit is not clear, but the observed higher progression (Beijersbergen et al., 1995; Kiess et al., anity of E2F-4 for p130 binding in vitro (Sardet et 1995; Hurford et al., 1997; Garriga et al., 1998). These al., 1995) suggests that the accumulation of p130 observations in conjunction with the opposing patterns displaces pRB from associating with E2F-4 complexes. of expression of p130 and p107 protein levels in a The fact that p1307/7 p1077/7 MEFs and lymphocytes number of cell growth and di€erentiation models show upregulated expression of some E2F-dependent (Kiess et al., 1995; Richon et al., 1997; Garriga et al., genes normally repressed in G0 indicates that pRB is 1998) strongly suggest that p107 is under the control of unable to substitute for all p130 mediated transcrip- p130, possibly through E2F-dependent transcriptional tional repression events. Conceivably, pRB might also regulation. However, serum starved p1307/7 mouse repress the expression of some genes in G0 through ®broblasts show abnormally elevated levels of p107 interaction with transcription factors other than E2F. protein, but, p107 mRNA remains unchanged, suggest- The biochemical events during G1 progression must ing that the e€ect of p130 on p107 expression is post- be very di€erent depending on whether we consider transcriptional at least in this cell type (Hurford et al., cells entering the cell cycle from G0 or cells after 1997). In contrast, serum-starved pRB7/7 ®broblasts do having exited mitosis in a continuously proliferating show elevation of p107 mRNA levels which suggests state. Particularly, G1 re-entrance from G0 involves an that pRB is a better candidate than p130 in conferring early stage where pre-existing p130/E2F-4 complexes the E2F-dependent transcriptional repression on p107 are still abundant and repress transcription from E2F- at least in these cells. Regarding other E2F-taget genes dependent promoters, whereas cells exiting mitosis into known to be upregulated during G1, it is dicult to G1 characteristically contain pRB/E2F complexes distinguish whether they are directly activated by (Mayol et al., 1996; Smith et al., 1996). These patterns disruption of p130/E2F-4 complexes or if subsequent are consistent with a di€erential role of pRB in the R expression of pRB-associated E2F members and point passage and of p130 in G0/G1 transitions (Mayol activation of pathways downstream of p130 are also et al., 1996). Quiescent cells do not express many cell involved. One should keep in mind that although the cycle regulators, which must be coordinately re- above mentioned genes are thought to be repressed synthesized during G1 for a proper cell cycle re- through E2F sites in G0, their expression timing during entrance. In this context, persistence of the cell cycle the cell cycle re-entry di€ers signi®cantly. In the case of exit-associated p130 phosphorylation that stabilize its cyclin A, for instance, it seems that its E2F dependent protein levels seem to be crucial in keeping E2F-4 as a expression in late G1 results from the sequential repressor during early G1. Further progression through repressive action of the three pocket proteins during the G1 phase leads to cyclin/CDK activation, and G1 progression. concomitant hyperphosphorylation of p130 to form 3 Recent experimental evidence indicate that the disrupts p130/E2F-4 complexes (Mayol et al., 1996), repressive action of E2F-pocket protein complexes on thereby releasing E2F-dependent transcriptional repres- cyclin gene expression is tightly regulated during G1 sion to allow for the expression of genes required for and it plays a major role in the R point transition. The passage through the R point and further cell cycle G1 arrest mediated by a pRB phosphorylation mutant progression. constitutively active in the binding to E2F allows the E2F-1 and E2F-2 are under E2F-dependent tran- expression of cyclins D and E, but not cyclin A scriptional control during the cell cycle, being not (Knudsen et al., 1998). Since p130 and p107 display expressed in resting cells and expressed in mid G1 hyperphosphorylation under this G1 arrest condition, coinciding with cyclin/CDK activation (Hsiao et al., these results suggest that the G1 phase has progressed 1994; Johnson et al., 1994; Neuman et al., 1994; to a pRB-sensitive critical point downstream of cyclin The pRB family in negative cell growth control XGranÄa et al 3373 D and E expression and activation of their associated 1998; Ferreira et al., 1998; Luo et al., 1998; Magnaghi- kinase activity. The fact that cyclin E expression is Jaulin et al., 1998) and the other two family members, una€ected by the pRB phosphorylation mutant p107 and p130 (Ferreira et al., 1998) to recruit histone suggests that E2F-dependent transcriptional activation deacetylase 1 (HDAC-1) to E2F site-containing of the cyclin E gene during G1 takes place after promoters (Figure 3a). phosphorylation of p130. Importantly, increasing the Histone deacetylases are enzymes that mediate the levels of cyclin E in these cells bypasses the G1 arrest removal of acetyl groups from core and other imposed by the pRB mutant, or by the CDK inhibitor proteins involved in transcription. This causes reorga- p16 (Alevizopoulos et al., 1997; Lukas et al., 1997), nization of that impairs the access of and allows S phase entrance, suggesting that over- transcription factors to DNA and thereby inhibits expression of cyclin E can activate pathways of passage transcription. Recently it has been shown that a through the R point independent or downstream of deacetylase activity associates with pRB both in vitro pRB/E2F control. With respect to cyclin A, its timing and in vivo. This associated activity is catalyzed by the of expression beginning in late G1 and its dependence HDAC-1 protein, which associates with pRB. pRB on cyclin E activation (Zerfass-Thome et al., 1997) interacts, through its A/B pocket domain, with an suggest that cyclin A expression is secondary to the R LXCXE-like motif (IACEE) located in the C-terminus point passage. In this context, it has been suggested of HDAC-1. The C-terminus of pRB is dispensable for that multimeric complexes containing p107/E2F and such interaction (Magnaghi-Jaulin et al., 1998), which cyclin E/CDK2 are targeted to the cyclin A gene is in agreement with the fact that the A/B pocket is promoter and that CDK2 activity is required for cyclin both necessary and sucient for the transcriptional A promoter activation in late G1 (Zerfass-Thome et repression activity of pRB (Johnson and Schneider- al., 1997; Schulze et al., 1998). Ectopic expression of Broussard, 1998). Moreover, the pRB/HDAC-1 bind- cyclin E allowed for the expression of cyclin A in the ing can be competed by peptides containing the presence of the pRB phosphorylation mutant (active LXCXE motif of viral proteins known to interact pRB), but the cells arrested during S phase progression with pRB, suggesting that those viral proteins may (Knudsen et al., 1998). Thus, these results suggest that release the pRB transcriptional repression by disrupt- phosphorylation of pRB after the G1/S transition is ing pRB/HDAC-1 complexes (Brehm et al., 1998; required. Keeping pRB, and perhaps other pocket Magnaghi-Jaulin et al., 1998). The notion that proteins, in its hyperphosphorylated state should repression of genes containing E2F sites by pRB is potentially allow for the continued expression of mediated by HDAC-1 is based on the following pieces certain genes required in S phase. of evidence: (i) E2F is not able to associate with Finally, recent experiments suggest that, once post- HDAC-1 in the absence of pRB, suggesting that pRB restriction point commitment has taken place, pocket tethers HDAC1 to E2F; (ii) in transient cotransfection proteins exert a direct role in modulating DNA experiments using a luciferase reporter gene under the replication during S phase. As seen earlier, over- control of an E2F promoter and expression vectors for expression of cyclin E bypasses a G1 arrest induced E2F, pRB and HDAC-1, expression of HDAC-1 by a constitutively active pRB mutant and results in increased E2F-mediated repression by pRB, whereas the entrance into S phase. However, cells do not in the absence of pRB, HDAC-1 has no e€ect on the complete S phase, which reveals an inhibitory function E2F promoter and (iii) this inhibitory e€ect was also of pRB on DNA replication (Knudsen et al., 1998). In demonstrated using a stably integrated reporter gene this respect, modulation of pRB/E2F and p107/E2F (Brehm et al., 1998), which best resembles the real complex levels during S phase might have a role in architecture of a gene promoter in its chromosomal controlling the expression of certain genes required for context. Since HDAC-1 enhances repression by pRB in DNA replication. Moreover, a G2 phase block induced both a genomic environment and in non-integrated by the CDK inhibitor p21 is accompanied by DNA plasmids, it seems that deacetylation events may also endo-reduplication in the absence of functional pRB occur in non-chromatin proteins. Although these data suggesting that suppression of DNA synthesis after S indicate that pRB uses deacetylase activity to repress phase completion is, at least partially, controlled by E2F, TSA (a deacetylase inhibitor) does not completely pRB in G2 arrested cells (Niculescu et al., 1998). release repression, suggesting that pRB, and by Interestingly, direct binding of pRB and p130 to extension other pocket proteins, use additional MCM7 protein, a DNA replication licensing factor, mechanisms to repress E2F-dependent promoters. has recently suggested that pocket proteins may Both, p107 and p130, have recently been demonstrated directly regulate the activity of these complexes by to interact with and repress E2F-4 activity through a direct protein-protein associations (Sterner et al., 1998). histone deacetylase. Thus, it seems that p107 and p130 Further studies, however, are needed to ascertain the also tether HDAC-1 or a related deacetylase to E2F functional extent of pocket protein interactions with promoters, indicating that the three pocket proteins the DNA replication machinery. share a similar model of E2F-mediated repression by recruiting HDAC-1 (Ferreira et al., 1998). Histone deacetylase Transcriptional repression of RNA polymerases I and III As discussed earlier, pocket proteins actively repress by pRB the transcription of E2F dependent genes. This occurs, at least in part, because of the ability of di€erent E2F/ pRB inhibits transcription by both RNA pol I and DP complexes to tether pocket proteins to the E2F RNA pol III. RNA pol I is responsible for the sites contained in responsive promoters. Repression transcription of the large ribosomal subunit (rRNA), appears to be due to the ability of pRB (Brehm et al., while RNA pol III is involved in the transcription of The pRB family in negative cell growth control XGranÄa et al 3374

Figure 3 Inhibition of the three RNA polymerases by pocket proteins. (a) Pocket proteins repress RNA pol II mediated transcription of E2F regulated genes. Repression is mediated, at least in part, by the ability of the pocket proteins to recruit histone deacetylase 1 (HDAC-1) to E2F site-containing promoters (see Figure 2a). (b) pRB represses the transcription mediated by RNA pol I. RNA pol I transcription is stimulated by UBF. When pRB binds to UBF, this transcriptional factor is unable to bind to the DNA and thus rRNA transcription is impaired. (c) pRB represses the expression of genes transcribed by RNA pol III. RNA pol III transcription requires the general transcription factor TFIIIB. pRB interacts and inactivates TFIIIB preventing activation of RNA pol III

the small rRNA subunit (5S rRNA) and tRNA. By UBF to the rDNA promoter is impaired (Voit et al., limiting the production of rRNA and tRNA, pRB 1997). might repress the level of protein synthesis, which in On the other hand, several lines of evidence turn would result in suppression of cell growth suggested that pRB also inhibits RNA pol III. The (reviewed by White, 1997). synthesis of RNA pol III products is inhibited when TPA-mediated di€erentiation of human monocyte- pRB is overexpressed in transfected cells or added in a like U937 cells correlates with downregulation of reconstructed in vitro system. When comparing two rRNA synthesis and results in translocation of pRB cell lines, which di€er in their pRB to the nucleolus. Since the nucleolus is the site of status, the activity of RNA pol III is much higher in RNA pol I transcription, these data suggest that pRB pRB de®cient SAOS-2 cells than in pRB wild type U-2 could mediate inhibition of transcription of rRNA OS cells. More convincing evidence comes from genes by RNA pol I (Cavanaugh et al., 1995). In measurement of RNA pol III activity in mouse support of this hypothesis, pRB has been found to embryonic ®broblasts (MEFs) obtained from pRB7/7 associate with UBF (Upstream Binding Factor), a mouse embryos. In pRB7/7 MEFs, RNA pol III transcription factor that, although not required for activity is four to ®ve times higher than in control basal rDNA transcription, stimulates RNA pol I ®broblasts (White et al., 1996). Since pRB is able to transcription by helping assembly of the initiation inhibit the transcription from three di€erent types of complex (Figure 3b). In this regard, it has been RNA pol III promoters, it was postulated that pRB shown that UBF/pRB interaction increases during could mediate this inhibition through a general U937 di€erentiation (Cavanaugh et al., 1995). More- transcription factor. TFIIIB, which directs RNA pol over, repression of UBF-stimulated RNA pol I III to the start site, seems to be such factor. TFIIIB transcription requires the C-terminus domain of and pRB co-purify during multiple diverse chromato- pRB, being the N-terminus dispensable. Interest- graphic steps and a physical interaction between pRB ingly, mutations on the A/B pocket do not seem to and TFIIIB was demonstrated by immunoprecipitation a€ect the ability of pRB of interacting with UBF. As and recombinant-protein pull-down experiments. pRB a result of pRB association with UBF, the binding of might disrupt TFIIIB function by mimicking two of its The pRB family in negative cell growth control XGranÄa et al 3375 subunits, TBP and BRF (Figure 3c), with which it al., 1997) or they act as genuine CKIs (Castano et al., shares some homology (Larminie et al., 1997). Whether 1998). In support of the later, low levels of ectopic p107 and or p130 regulate RNA pol III is not known. p107 in SAOS-2 cells led to a decrease in CDK2 The fact that RNA pol III activity is increased in activity, which occurred without changes in the levels pRB7/7 MEFs (White et al., 1996), demonstrates that of CDK2 and cyclins A and E, apparently ruling out neither p107 nor p130 compensate for this function of E2F-mediated repression of cyclin A and cyclin E pRB in its absence. This result indicates that either genes. In keeping with this notion, levels of CDK2 p107 and p130 do not regulate RNA pol III or that the activity were found to be increased in p1077/7 and levels of these proteins are not sucient to repress p1077/7 p1307/7 MEFs (Castano et al., 1998). Never- RNA pol III in the absence of pRB. theless, the possibility that these in vivo e€ects are The implications of the regulation of RNA pol I and indirect has not been ruled out. III by pRB in cancer are clear from the following facts: Although the biochemical grounds supporting a (i) the domains of pRB required for growth suppres- function for p107 and p130 as CKIs are sound, one sion are also required for inhibition of the two should keep in mind the relative abundance of these polymerases by pRB and (ii) naturally occurring proteins and the complexes that contain them in mutations that render pRB inactive as tumor di€erent physiological cellular instances. This is suppressor also compromise its ability to inhibit the particularly important for p130 since its levels drop activity of RNA pol I and III (White, 1997). In dramatically as cells progress through S phase and agreement with these results, the activities of RNA pols mitosis. Thus, the relative levels of p130/cyclin (E or I and III are found elevated in murine tumors and are A)/CDK2 complexes compared to p107/cyclin (E or elevated by transforming agents (reviewed by White, A)/CDK2 complexes and/or other cyclin (E or A)/ 1997) CDK2 complexes must be very low. One might argue that when cells exit the cell cycle, p130 levels rise sharply leading p130 to function as a CKI. However, p107 and p130 as CDK inhibitors accumulation of p130 only occurs in the G1 phase Two short amino acid motifs have been identi®ed in before the G1/G0 transition point, at a time when the p107 and p130, which are required for binding of p107 CDK2 associated cyclin E and cyclin A kinase and p130 to cyclin E/CDK2 or cyclin A/CDK2 activities are already low due to normal cell cycle complexes (Adams et al., 1996; Lacy and Whyte, dependent regulation (Mayol et al., 1996). Moreover, 1997; Woo et al., 1997; CastanÄ o et al., 1998). Similar we should also consider that a number of cellular short amino acid motifs are found in the CKIs (p21, pathways, which lead to cell cycle exit, result in p27, p57) and E2Fs (E2F-1, -2, -3) that bind cyclins E accumulation and binding of CKIs to their correspond- and A. One of these motifs is present in the spacer ing CDK or cyclin/CDK targets, but no increase of region of p130 and p107, but is not found in the highly p107 and/or p130 bound to cyclin/CDK2 complexes divergent spacer region of pRB (Adams et al., 1996; has been as to yet reported. Indeed, binding of p21 and Lacy and Whyte, 1997). In agreement with this, pRB p107 or p130 to cyclin/CDK2 complexes is mutually does not bind to cyclins E and A. The other motif is exclusive, and upregulation of p21 leads to abrogation found at the amino terminus (Castano et al., 1998). of the p130 or p107 binding to cyclin/CDK2 (Zhu et Mutations in either one motif are sucient to abolish al., 1995b; Shiyanov et al., 1996). the interaction of p107 and p130 with these cyclins (Adams et al., 1996; Lacy and Whyte, 1997; Castano et Control of di€erentiation through regulation of cell al., 1998). Since the ®rst binding motif identi®ed in the type-speci®c gene expression spacer region of p107 and p130 shared identity with proteins that are either CDK substrates or CKIs Pocket proteins are important regulators of certain (Adams et al., 1996), it was suggested that CKIs developmental and di€erentiation processes (for a could function at least in part by blocking the access of recent extensive review see Yee et al., 1998). In vitro substrates to the kinase catalytic subunit (Adams et al., cell di€erentiation is typically characterized by the 1996). The notion that p107 and p130 could act as activation of mechanisms of growth arrest that lead to CKIs was proposed based on the fact that the ability cell cycle exit before genetic programs of di€erentiation of p107 and p130 to suppress growth depends, at least are turned on. Particularly, regardless of di€erentiation in part, on their ability to inhibit CDK2 activity (Zhu inducers, negative cell cycle regulatory pathways et al., 1995a, b; Deluca et al., 1997; Woo et al., 1997; converge in the downregulation of E2F-dependent Castano et al., 1998). In this context, it has been shown gene expression by formation of pocket protein/E2F that p107 exhibits an inhibitory constant (Ki) towards repressor complexes in a number of in vitro cyclin E/CDK2 and cyclin A/CDK2 comparable to di€erentiating cell types, including di€erentiation of that of the CKI p21. In agreement with these results, skeletal muscle cells (Corbeil et al., 1995; Halevy et al., puri®ed recombinant p107/cyclin/CDK2 or p130/ 1995; Kiess et al., 1995; Shin et al., 1995), neuronal cyclin/CDK2 complexes possess lower kinase activity cells (Corbeil et al., 1995), melanoma cells (Jiang et al., towards exogenous substrates such as histone H1 than 1995), HL60 cells (Ikeda et al., 1996; Smith et al., 1996) single cyclin/CDK2 complexes (Woo et al., 1997). and 3T3-L1 adipocytes (Richon et al., 1997) as well as Since p107 and p130 are likely in vivo substrates of in vivo re-di€erentiation of mouse hepatocytes after cyclin/CDK2 complexes, the results obtained by liver (Garriga et al., 1998) and retina di€erent groups summarized above have led to distinct di€erentiation (Kastner et al., 1998; Rampalli et al., interpretations. These include that: p107 and p130 are 1998). As discussed earlier, most of the E2F-mediated preferential substrates (Adams et al., 1995), they transcriptional repression in this G0 condition seems to change the substrate speci®city of CDK2 (Hauser et be typically exerted by p130/E2F-4 complexes. On the The pRB family in negative cell growth control XGranÄa et al 3376 other hand, E2F-dependent transcriptional regulation (Iavarone et al., 1994), and with the thyroid hormone has never been shown to take place in genes involved -coactivator Trip230 (Chang et al., 1997) have in cell di€erentiation. This suggests that the role of a less clear role in di€erentiation but may be signi®cant pocket proteins in the control of di€erentiation in certain homeotic and tissue-speci®c growth pro- programs involves transcriptional regulation of gene cesses. expression by pathways other than, or additional to, In some cases, di€erential ability of pocket proteins the ones used to stop the cell division cycle. to bind to some of these transcription factors Consistently, pRB mutants defective in E2F binding, correlates with a particular functional involvement and therefore, in inducing G1 arrest are still capable of in the cell di€erentiation process. Thus, although inducing di€erentiation-associated ¯at cell morphology p107 and/or p130 can partially substitute for pRB in osteosarcoma cells as well as transactivating absence in regulating gene expression by myogenic heterologous promoters from genes induced during transcription factors, they do not normally do so in di€erentiation (Sellers et al., 1998). wild type pRB cells and pRB is unique in regulating Experimental evidence implicate pocket proteins as late markers of muscle di€erentiation, such as myosin regulatory factors required in cell di€erentiation heavy chain expression (Novitch et al., 1996). processes. Firstly, pocket protein gene targeting in Consistent with this, pRB de®ciencies in mice lead mice has revealed developmental abnormalities in to defects in skeletal muscle development, among several tissues. Secondly, ectopic expression of pocket other tissues, while p107 and/or p130 de®ciencies proteins, and particularly of pRB, in certain cell types show no abnormalities in this tissue. The unique induces either morphological di€erentiation or tran- ability of pRB to complex with myogenic transcrip- scriptional activation of tissue-speci®c genes, for tion factors such as MyoD and in vivo, instance, the above mentioned ¯at cell morphology in may be an explanation for this di€erent functional osteosarcoma cells (Sellers et al., 1998), muscle or behavior. In other cases, di€erential binding abilities adipocyte di€erentiation by re-introduction of pocket among pocket proteins have just not been assessed. proteins in pRB7/7 ®broblasts (Gu et al., 1993; The interaction of pocket proteins with transcription Schneider et al., 1994; Chen et al., 1996a; Novitch et factors involved in di€erentiation takes place upon al., 1996), neuronal di€erentiation of PC12 cells cell cycle exit, thus indicating that hypophosphory- (Kranenburg et al., 1995), and keratinocyte differentia- lated forms of pocket proteins are involved. Since no tion markers in HaCaT cells (Paramio et al., 1998). cyclin/CDK activities target pocket proteins in Moreover, pRB anti-sense oligonucleotides block in di€erentiating cells, these associations may not be vitro muscle di€erentiation of C2 myoblasts (Kobaya- regulated by phosphorylation during the differentia- shi et al., 1998). Thirdly, pocket proteins, pRB being tion process. However, pocket protein hypophosphor- the family member most extensively analysed, have ylation may be a prerequisite, as exempli®ed by the been found to associate directly with a number of fact that MyoD is present in proliferating myoblasts proteins with tissue-speci®c activities (Table 1). Some but only binds to pRB when cells have exited the cell of these proteins are transcription factors involved in cycle (Gu et al., 1993). It remains to be analysed if the regulation of gene expression during di€erentiation the cell cycle exit-speci®c phosphorylation on p130 processes: (i) pRB, and p107 in pRB7/7 cells, associate has a role in the association of p130 with some of with MyoD and myogenin during skeletal muscle these transcription factors. di€erentiation and activate the expression of muscle Regulation of pocket protein levels do seem to play speci®c genes (Gu et al., 1993; Schneider et al., 1994; a role in the coordination of pocket protein interac- Novitch et al., 1996); (ii) muscle di€erentiation also tions during some di€erentiation processes. Thus, cell involves association of the transcription factors HBP1 cycle exit-associated accumulation of p130 results in with pRB and p130 (Lavender et al., 1997; Tevosian et abundant p130/E2F-4 complexes that repress transcrip- al., 1997) and p202 with pRB (Choubey and Lengyel, tion of cell cycle genes as a ®rst step in di€erentiation, 1995; Choubey and Gutterman, 1997; Datta et al., although pRB/E2F complexes have also been detected 1998); (iii) pRB binds to the related transcription in some cases (Corbeil et al., 1995; Halevy et al., 1995; factors C/EBP and NF-IL6 in adipocytes and Ikeda et al., 1996; Rampalli et al., 1998). The promotes adipocyte di€erentiation (Chen et al., abundance of these pocket protein/E2F complexes, 1996a,b); (iv) the nuclear matrix protein NRP/B, not only allows cell cycle exit, but also, may confer which is required for neuronal di€erentiation, forms reversibility to the di€erentiation process, at least in complexes with pRB (Wiggan et al., 1998); and (v) the early stages. While the involvement of pocket pRB binding to AP2 complexes activate the AP2- proteins in inducing di€erentiation steps rely on dependent transcription of E-cadherin, an intercellular punctual biochemical data in many cases, skeletal adhesion protein involved in cell polarity and muscle di€erentiation has been the most extensively di€erentiation of epithelial cells (Batsche et al., 1998). studied process in this respect, being currently clear Furthermore, and with uncertain tissue speci®city but that involves a cascade of transcriptional with a possible homeotic role, the three pocket proteins events tightly regulated by interdependent myogenic associate with paired-like homeodomain transcription transcription factors (reviewed in Molkentin and factors, including MHox, Chx10, Alx3, and Pax3 Olson, 1996; Rawls and Olson, 1997) and, at least in (Wiggan et al., 1998). In these studies, Pax3-dependent part, by the coordinated action of pRB and p130 transcription was shown to be repressed by pocket (reviewed in Yee et al., 1998). In this respect, pRB proteins. Finally, association of pRB with the related upregulation might trigger a switch from an early transcriptional co-activators BRG1 and hBRM reversible phase mediated by di€erentiation-repressing (Dunaief et al., 1994; Singh et al., 1995; Strober et factors, such as HBP1, to terminal di€erentiation (Shih al., 1996), with the transcriptional co-repressor Id2 et al., 1998). The pRB family in negative cell growth control XGranÄa et al 3377 The biochemistry and biology of pocket proteins tested development, chimeras partially composed of pRB7/7 by their knockouts cells in a p1077/7 animal context undergo retinal dysplasia already detectable during fetal development Pocket proteins do share a number of upstream (Robanus-Maandag et al., 1998) and pRB+/7 p1077/7 regulatory mechanisms and also seem to regulate a animals do so during adulthood (Lee et al., 1996), common subset of cellular targets. For instance, the suggesting that sporadic loss of the remaining pRB three proteins are hyperphosphorylated in mid-to-late generates these lesions in the absence of p107. G1 most likely by cyclin/CDK complexes. However, Since no other combination of disruption of pRB these common upstream pathways have profound family members exhibit this phenotype, it appears that di€erent e€ects on the fate of the three pocket either pRB, p107 or both are essential for normal proteins. Although pocket proteins share the ability development of mouse retina and p130 cannot of interacting with various common cellular proteins, a compensate for their loss (Lee et al., 1996). The number of interactions seem to be speci®c for each involvement of pRB in lens development was studied particular pocket protein. p107 and p130 are in pRB de®cient embryos and in transgenic mice structurally more closely related and, consistent with expressing papillomavirus E7 protein (which blocks this, there are a number of proteins or protein the function of the pRB family) in the developing lens. complexes that interact with them, which do not Inactivation of pRB function resulted in unchecked interact with pRB. These di€erences are likely to proliferation, inhibition of di€erentiation and apopto- re¯ect the di€erent cellular function of each pocket sis. Apoptosis was dependent on a functional , since protein, while the similarities suggest that all pocket pRB7/7 p537/7 embryos, transgenic mice expressing proteins might act coordinately to regulate a common papillomavirus E7 and E6 (which inactivates p53 set of cellular targets. In addition, we should also function) proteins or transgenic mice expressing the consider that some functions of pocket proteins might E7 protein in a p537/7 background did not exhibite be redundant, and that di€erent pocket proteins might apoptosis in lens. be capable of carrying out the same cellular function in In regard to tumor susceptibility in pocket protein- di€erent cell types de®cient mice, the phenotypes of adult animals have revealed that the tissue-range tumor suppressor function of pRB may be limited, since pRB+/7 mice Phenotypes of pRB family mutant mouse strains only develop thyroid and pituitary tumors. Interest- Gene targeting experiments in mice have begun to ingly, retinoblastomas, which primarily harbor pRB delineate clear di€erences among pocket proteins. mutations in humans, are never observed in the pRB+/7 However, these experiments have also indicated that animals (Jacks et al., 1992; Hu et al., 1994; in the absence of a particular pocket protein Morgenbesser et al., 1994), which has been attributed mechanisms of compensation by the remaining pocket to di€erences between human and mouse retina. proteins exist (for a more detailed review of these Pituitary tumors are composed of pRB7/7 cells studies see Mulligan and Jacks, 1998). indicating loss of the wild type allele. Conversely, The most severe phenotype observed from analysis introduction of a wild type pRB transgene in these of the mouse mutant strains lacking the function of a pRB+/7 mice completely prevents tumor formation pRB family member is caused by disruption of the RB (Riley et al., 1997). Tumor suppression by pRB can gene. Mice lacking functional pRB die during also be assessed using the pRB-de®cient chimeras embryogenesis with defects in haematopoiesis, neuro- which, in agreement with previous results, only genesis and defects in liver and lens development undergo pituitary tumorigenesis, being the tumors (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992; analyzed almost exclusively formed by pRB7/7 cells Morgenbesser et al., 1994). Although milder, similar (Maandag et al., 1994; Williams et al., 1994). This abnormalities were found in pRB chimeric mice indicates that the second mutational event found in the partially composed of pRB-de®cient cells, which are wild type-pRB allele of pituitary tumors from pRB+/7 rescued from the pRB-de®cient lethality and display mice (Jacks et al., 1992; Hu et al., 1994) is critical for extensive contribution of pRB7/7 cells in adult tissues tumor formation. Consistent with this, the age of (Maandag et al., 1994; Williams et al., 1994). These chimeric animals in which pituitary tumors were de- studies indicate that the function of pRB in certain cell tected was signi®cantly reduced compared to pRB+/7 types is essential for their normal development and that animals. On the other hand, the fact that pRB gene neither p107 nor p130 can compensate for pRB targeting in mice only provokes thyroid and pituitary absence. In contrast, disruption of either p107 or tumors does not rule out that pRB loss can also p130 genes leads to no detectable developmental defect contribute to in other tissues. Thus, in a (Cobrinik et al., 1996; Lee et al., 1996). Interestingly, pRB-de®cient setting, the time before mice die from double p1077/7 p1307/7 mice die shortly after birth pituitary cancer may be too short to allow for because of breathing diculties, which arise from a additional mutations in other genes, which are known defect in endochondral bone formation. The defect in to be required in the multi-step character of endochondral bone formation a€ects long bones and carcinogenesis in many tissues. In support of this, ribs, and is due to excessive chondrocyte proliferation transgenic mice expressing E7 in lens displayed tumors (Cobrinik et al., 1996). These results indicate that only in the absence of functional p53 (Howes et al., either p107 or p130 or both are essential for cell cycle 1994; Morgenbesser et al., 1994; Pan and Griep, 1994). exit in this cell type and that this function cannot be Moreover, induction of tumors in the brain choroid compensated by pRB or by any other as to yet plexus epithelium demonstrated a cooperation in unknown member of this family. Moreover, in contrast tumorigenesis by inactivation of p53 and the pRB to pRB+/7 animals, which show normal retinal family (Symonds et al., 1994). Therefore, the murine The pRB family in negative cell growth control XGranÄa et al 3378 pituitary gland, similar to the retina in humans, might erythropoiesis, skeletal muscle and nervous system while be a tissue highly susceptible to cell transformation the wild type pRB transgene allows normal development when pRB function is impaired, whereas other tissues of these tissues (Riley et al., 1997). Moreover, this pRB may require further mutational alterations, such as loss mutant transgene does not prevent, as wild type pRB of p53 function. Similarly, the above mentioned retinal does, the appearance of thyroid tumors in pRB+/7 mice dysplasia in pRB-chimeric, p1077/7 mouse fetuses is (Riley et al., 1997). These studies suggest that at least followed by the formation of retinoblastomas in some of the e€ects of pRB absence in mice are not the adulthood (Robanus-Maandag et al., 1998), suggest- result of an impairment of the pRB targeting function ing again that additional alterations are limiting for the on E2F-1. of these pRB/p107-de®cient retinal Double mutant pRB7/7 E2F-17/7 embryos exhibit pre-neoplastic lesions. Since neither pRB+/7 mice nor reduced apoptosis and S phase entry in certain tissues, pRB-de®cient chimeric animals display retinal tumors such as lens and CNS, when compared with their (Jacks et al., 1992; Hu et al., 1994; Maandag et al., counterpart pRB7/7 tissues, indicating that pRB 1994; Williams et al., 1994), p107 might have a role as normally represses E2F-1-mediated S phase induction a tumor suppressor in the retina. However, the and apoptosis in these tissues. The involvement of p53 functional implications of p107 in carcinogenesis are in these apoptotic scenarios was indicated by the ®nding still unclear because pituitary tumors are abrogated in that p53 DNA-binding activity and the expression of a pRB+/7 p1077/7 compared to pRB+/7 mice (Lee et al., p53 target gene, the p21CIP1 gene, are reduced in the 1996). Indeed, in contrast to pRB, neither heterozygous pRB7/7 E2F-17/7 double mutant and wild type brains, nor homozygous deletions of p107, p130 or both in while they are increased in pRB7/7 brains (Pan et al., mice lead to tumor formation in any tissue (Cobrinik 1998; Tsai et al., 1998). Similar conclusions were et al., 1996; Lee et al., 1996), which is in agreement reached by using a transgenic model in which with the fact that these genes have rarely been found expression of a N-terminal fragment of T antigen is mutated in human cancer. Nevertheless, in support of a targeted to the choroid plexus. Expression of this T role for p130 and p107 as tumor suppressors, it has antigen (T121) mutant blocks the pRB family, but does been shown that a growth advantage conferred by not block p53. Aggressive epithelial brain tumors are SV40 large T-antigen in MEFs requires inactivation of induced by this T antigen mutant in a p537/7 back- both p130 and p107, a feature that might contribute to ground. In a p53+/+ background, p53 target genes are T-antigen-mediated cell transformation (Christensen upregulated instead and apoptosis occurs, resulting in and Imperiale, 1995; Zalvide and DeCaprio, 1995; tumor growth attenuation (Symonds et al., 1994). Stubdal et al., 1997). Interestingly, lack of E2F-1 in the T121 transgenic mice results in reduction of apoptosis and this correlates with absence of upregulation of p53-regulated genes. E2F-1 is a downstream target of pRB in E2F-1 mediated However, no tumors generate because E2F-1 absence induction of S phase, apoptosis and tumorigenesis also impairs S phase induction (Pan et al., 1998; Tsai et As we have discussed in previous sections, pRB al., 1998). Altogether, these ®ndings suggest a model in negative function on cell proliferation is thought to which pRB restricts induction of both S phase and p53- be mediated, at least in part, by repression of members dependent apoptosis by E2F-1. of the E2F family of transcription factors. Among E2F family members, E2F-1 is speci®cally targeted by pRB, Cellular and molecular consequences of disruption of the but not by p107 or p130. The role of E2F-1 as a function of pRB family members downstream target of pRB during tumorigenesis is starting to be understood. In pRB+/7 E2F-17/7 mice The e€ects of disrupting the function of each pRB the frequency of thyroid and pituitary tumors is family member as well as their combinations have been reduced compared to the frequency in pRB+/7 mice studied in detail in mouse embryonic ®broblasts harboring wild type E2F-1, which demonstrates that (MEFs) and, in the case of p130 and/or p107, also in E2F-1 is involved in the development of tumors that peripheral T lymphocytes. Firstly, these experiments arise in pRB+/7 mice (Yamasaki et al., 1998). have shown both di€erences in the time period The role of E2F-1 as a downstream target of pRB in required to reach S phase upon serum re-stimulation cell cycle control and apoptosis in vivo has been studied of MEFs and di€erences in the patterns of expression during embryonic development and in epithelial brain of a number of E2F-regulated genes. Secondly, these tumors (Pan et al., 1998; Tsai et al., 1998). Double experiments have also shown functional compensation pRB7/7 /7 embryos die by E17, later than pRB7/7 among pocket proteins (Almasan et al., 1995; Lukas et embryos which die between E14 and E15, with anemia al., 1995a; Herrera et al., 1996; Hurford et al., 1997; and defective muscle and lung development. Since E2F- Mulligan et al., 1998). 17/7 mice are viable (Field et al., 1996; Yamasaki et al., In respect to the di€erences in the expression of 1996), these results indicate that although E2F-1 E2F-regulated genes, MEFs lacking functional pRB, function is required for some of the defects observed but not p107 or p130, exhibit derepression of a subset in pRB7/7 embryos, it is not the sole essential target of of E2F-target genes in G0/G1, including the cyclin E pRB during mouse embryonic development. Other and p107 genes. Compensation among pocket proteins essential targets of pRB might include other E2F is suggested by the fact that only MEFs lacking both family members and/or transcription factors involved p107 and p130 exhibit deregulation of a di€erent in cell di€erentiation. In this context, transgenic pRB7/7 subset of E2F-target genes in G0/G1, including B-, fetuses expressing an N-terminal truncated pRB cdc2, E2F-1, TS, ribonucleotide reductase subunit M2 mutant, which retains the ability to bind to E2F, have (RRM2), cyclin A and DHFR genes (Hurford et al., a delayed prenatal death but still show abnormalities in 1997). The fact that p130 is expressed at high levels in The pRB family in negative cell growth control XGranÄa et al 3379 quiescent wild type cells and is the major pocket For instance, the cell cycle exit pattern of pocket protein associated with E2F in these cells, suggests that proteins triggers a di€erentiation program only when lack of p130 is compensated by p107, but in the transcription factors and other mediators required for absence of both proteins pRB cannot repress at least that program are present. Such is the case when some of the E2F-target genes normally repressed by expression of MyoD or C/EBP transcription factors p130. This is further supported by the ®nding that pRB only promote di€erentiation in pRB7/7 ®broblasts if and p130/p107 seem to regulate di€erent E2F-target pRB is re-introduced in the cells. This reasoning could genes. be extended to many other scenarios. Therefore, pocket Analysis of the expression of E2F-target genes in proteins could be de®ned as competence factors which, p1077/7 p1307/7 T lymphocytes exhibited similar as discussed in this review, will allow a number of elevated expression of B-myb, cdc2, and diverse cell growth and di€erentiation transitions. RRM2 mRNAs when compared with p107+/7 p1307/7 It is assumed that a universal mechanism of cell and p1077/7 p130+/7 T lymphocytes (Mulligan et al., cycle exit involves transcriptional silencing of E2F- 1998). In murine T lymphocytes lacking p130, p107 responsive genes by pocket proteins. Thus, a multitude levels are elevated, and the E2F DNA binding activity of negative growth signals converge by di€erent detected contains p107 instead of the absent p130. In mechanisms in the inactivation of cyclin/CDK activ- p1077/7 p1307/7 peripheral T lymphocytes higher levels ities leading to the hypophosphorylation of pocket of free E2F are detected than in p1307/7 cells, but most proteins (reviewed by GranÄ a and Reddy, 1995; Mayol of the remaining E2F DNA binding activity contains and GranÄ a, 1998; Yee et al., 1998). In this scenario, pRB. Interestingly, a new E2F complex containing a complex formation with E2F is mostly regulated by the protein di€erent from the known members of the pRB phosphorylation state of pocket proteins. Silencing of family seems to be induced in the absence of p107 and genes involved in cell cycle progression will thus be p130. A similar complex was also detected in p1077/7 determined by the active repression of pocket protein/ p1307/7 MEFs (Hurford et al., 1997; Mulligan et al., E2F complexes or by the absence of free, transcrip- 1998). Thus, clear compensation mechanisms exist, tionally-activating E2F proteins due to pocket protein such that particular functions of each pocket protein sequestration. Since cell growth regulatory pathways in are preserved in their absence. p130 and p107 seem to G1 are complex and often involve feedback regulatory compensate for each other quite eciently, and this loops, determining pocket protein cooperativity during might explain the lack of abnormalities in the p1077/7 cell cycle exit is dicult. In this context, the presence of and p1307/7 animals. This conclusion is in agreement pRB/E2F and p107/E2F complexes in proliferating with the higher structural similarity between these two cells, but not p130/E2F-4, suggests that the involve- proteins when compared to pRB, and the fact that ment of E2F in the passage through the R point is p107 and p130 interact with a common subset of regulated by pRB and/or p107 rather than by p130. It proteins, which do not interact with pRB or exhibit is still unclear if the cell cycle exit-speci®c phosphor- lower anity for pRB. The much severe phenotypes ylation of p130, leading to its accumulation in the form seen in pRB7/7 mice, indicate that a number of of p130/E2F-4 complexes, is a triggering event of cell essential functions of pRB, cannot be performed by cycle exit or a consequence of it. either p107 or p130. This might be due to the inability Once in quiescence, p130/E2F complexes persist and, of p107 or p130 to interact with a number of pRB in fact, are typically found in many di€erentiated adult targets such as E2F family members 1, 2 and 3, as well tissues. During cell di€erentiation, keeping E2F- as other transcription factors. Alternatively, the levels dependent cell cycle genes silent seems to be a pre- of expression of p130 or p107 in the a€ected tissues requisite for the expression of di€erentiation genes might not be sucient to substitute for the absence of (Rao et al., 1994; Skapek et al., 1995; Wang et al., pRB. The higher compensatory eciency between p130 1996; Guo and Walsh, 1997). Considering that the and p107, together with pRB ability to compensate at repressor activity of p130/E2F-4 complexes is possibly least partially for p107/p130 loss might also explain a requirement for G1 re-entrance from G0, their the absence of tumors in p107+/7, p130+/7, p107+/7 presence in quiescent cells may also have the p1307/7 and p1077/7 p130+/7 animals. signi®cance of conferring reversibility to the cell cycle exit program. In the context of cell di€erentiation, p130/E2F complexes have been assigned to play a role Signi®cance of the regulation of pocket proteins in the in an early, di€erentiation-independent phase of cell negative control of cell growth cycle exit (Shin et al., 1995; Richon et al., 1997; Puri et al., 1998). This role may be speci®c to p130, or at least The wide range of pocket protein interactions with normally carried out by p130, because pRB absence, several cellular proteins (Table 1), together with the conversely, leads to a defect in terminal di€erentiation ubiquitous nature of some mechanisms of pocket that causes cell cycle exit reversibility (Schneider et al., protein action, such as modulation of E2F transcrip- 1994; Novitch et al., 1996; Zacksenhaus et al., 1996). tion factors, have indicated that the role of pocket The existence of certain tissue-speci®c transcription proteins is not restricted to cell types or to cell factors involved in negative regulation of gene functions. Moreover, diverse aspects of pocket protein expression during di€erentiation, such as HBP1 and function are tightly regulated primarily by two simple p202 in muscle and CHOP in adipocytes, is in support mechanisms: phosphorylation status and protein levels. of this reversible early phase (reviewed in Yee et al., The conserved mechanisms of pocket protein regula- 1998). In particular, HBP1 requires binding to pocket tion in diverse cell types and growth instances is in proteins for its negative function in muscle differentia- contrast to the dramatically di€erent consequences that tion and, in fact, increasing levels of pRB reverse this seem to depend on the epigenetic context of the cell. e€ect and allow myogenic gene expression (Shih et al., The pRB family in negative cell growth control XGranÄa et al 3380 1998). These data have led to propose that upregula- The fact that p130/E2F-4 complexes repress the tion of pRB protein levels, which is observed during in expression of the pRB-associated E2Fs (E2F-1 and vitro di€erentiation of skeletal muscle cells, would E2F-2) also plays a role in conferring order to the trigger the transition from an early reversible phase to coordinated action of pocket proteins during G1. In terminal di€erentiation (reviewed in Yee et al., 1998). this case, pRB action on E2F-dependent gene The transcription factors HBP-1, p202 and CHOP not expression would be dependent on the previous action only repress di€erentiation gene expression but also of p130. Finally, by extension, p107 expression in mid induce cell cycle exit when overexpressed (Barone et G1 will also be dependent on this activating cascade al., 1994; Tevosian et al., 1997; Datta et al., 1998), and since it is repressed by both p130 and pRB. Therefore, in the case of p202, direct inhibition of either E2F-1 or regulation of pocket protein phosphorylation status E2F-4 transcriptional activity has been reported and levels during G1 integrates growth signal (Choubey and Lengyel, 1995; Choubey and Gutter- transduction pathways by coordinating the interaction man, 1997). These data indicate that these transcription of pocket proteins with E2F and other transcription factors have a dual role in the early reversible phase of factors (see Table 1). This culminates in the passage di€erentiation: induction of cell cycle exit, perhaps in through the R point and in further progression cooperation with pocket protein/E2F repressor com- through S phase. plexes, and delay of di€erentiation gene expression Precise details on how pocket proteins regulate cell until terminal di€erentiation inducers (such as pRB) growth and di€erentiation processes are thus starting are activated. However, the biological signi®cance of a to be understood. Although the coordination in the pocket protein-dependent reversible period during interactions with cellular proteins during cell growth di€erentiation where cells exercise an option to situations is yet to be precisely grasped in many undergo di€erentiation is still unclear. aspects, it seems clear that the presence of a particular Upon cell cycle re-entry, it is striking that only p130/ phosphorylated pocket protein form in a given cell E2F complexes are found in the early, pre-R point type, growth stage, and cellular compartment has passage phase. It is conceivable that E2F transcrip- determinant consequences in the physiology of the tional repression during this stage underlies a complex cell. The above discussed cellular processes seem to be network of growth factor transduction pathways that dependent on the ability of pocket proteins to need to be coordinately activated for a proper cell cycle coordinate a multitude of regulatory processes acting re-entrance (reviewed in Mayol and GranÄ a, 1998). As as competence factors to allow or impede certain cell an example, one of the genes thought to be repressed growth and/or di€erentiation transitions. by p130/E2F-4 in early G1 is the E2F-1 gene. Forced overexpression of E2F-1 in serum-starved cells, that is, in the absence of activation of G1 pathways upstream of E2F-1, leads to an improper S phase entrance truncated by the induction of apoptosis (Johnson et al., Note added in proof 1993; Qin et al., 1994; Shan and Lee, 1994; Wu and After this review article was submitted to the publishers Levine, 1994; DeGregori et al., 1995; Kowalik et al., two papers were published describing the generation of 1995). Interestingly, induction of apoptosis by E2F-1 is p1307/7 and p1077/7 mutant mice in enriched Balb/cJ separable from its transcriptional transactivation backgrounds (LeCourter et al., 1998a: Development 125, capacity but dependent on DNA binding (Phillips et 4669 ± 4679; LeCourter et al., 1998b: Mol. Cell. Biol. 18, al., 1997). A transactivation domain-defective E2F 7455 ± 7465). Balb/cJ mice lacking p130 died between mutant relieves, probably by competition for DNA embryonic days 11 and 13 due to developmental abnorm- binding sites, the transcriptional repressor activity of alities, which in contrast with the apparent lack of p130/E2F-4 complexes in quiescent cells. Altogether abnormalities of the previously obtained mutant animals generated in a di€erent genetic background. Balb/cJ mice these results suggest that suppression of E2F-mediated lacking p107 are viable but exhibit impaired growth. transcriptional repression accounts for, at least in part, Interestingly both mutant animals revert to a wild type S phase induced apoptosis by E2F-1. In this context, phenotype when crossed with C57BL/6J mice, suggesting p130/E2F-4-mediated repression of genes like the E2F- the existence of modi®er genes that have epistatic relation- 1 gene may perform the function of keeping key ships with p130 and p107. A second study describing the regulators of R point passage silent until a number of association of HDAC1 with p130 has also been published G1 regulatory pathways have been activated and (Stiegler et al., 1998: Cancer Res. 58, 5049 ± 5052). integrated into a single R point passage decision. In the absence of p130-mediated transcriptional repres- sion, that is, in post-mitotic G1 proliferating cells, pRB Acknowledgements assumes the role of controlling E2F function in G1 by We thank Sushil Rane, Ana Limo nandMatildeParrenÄ o regulation of E2F-3, but not other E2F family for critical reading of the manuscript and suggestions. We members, at least in Ref 52 cells (Leone et al., 1998). apologize to colleagues whose work has not been cited, or The consequences, in terms of gene expression, of cited indirectly through other articles, due to space limitations. XM and JG were supported by fellowships whether E2F is regulated by p130 or by pRB during from Direccio n General de Investigacio nCientõ ®ca y early G1 are not known. Moreover, p130/E2F Te cnica (Ministerio de Educacio n y Cultura, Spain). Work repressor complexes have the responsibility of main- in XG laboratory was supported by a grant from the taining a pool of E2F proteins ready to trigger E2F- National Institute of General Medical Sciences, NIH dependent transcription upon cyclin/CDK activation. (GM54894). The pRB family in negative cell growth control XGranÄa et al 3381 References

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