The retinoblastoma family of proteins, also known as pocket proteins, includes the product of the retinoblas- toma susceptibility gene and the functionally and.
Oncogene (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 retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth Xavier GranÄa*,1, Judit Garriga1 and Xavier Mayol2 1
Fels Institute for Cancer 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 proteins, also known as pocket proteins, includes the product of the retinoblastoma susceptibility gene and the functionally and structurally related proteins p107 and p130. Pocket proteins control growth processes in many cell types, and this has been linked to the ability of pocket proteins to interact with a multitude of cellular proteins that regulate gene expression at various levels. By regulating gene expression, pocket proteins control cell cycle progression, cell cycle entry and exit, cell dierentiation and apoptosis. This review will focus on the mechanisms of regulation of pocket proteins and how modulation of pocket protein levels and phosphorylation status regulate association with their cellular targets. The coordinated regulation of pocket proteins provides the cells with a competence mechanism for passage through certain cell growth and dierentiation transitions. Keywords: CDK; CKI; transcriptional regulation; cyclins; cell cycle
Introduction Inactivation of both copies of the retinoblastoma gene is associated with the development of retinoblastomas in humans. In addition, other types of tumors also exhibit functional inactivation of the retinoblastoma gene product, pRB, together with defects in other genes. Indeed, deregulation of the pRB pathway is very common in human cancer (for review see Weinberg, 1995; Mulligan and Jacks, 1998). These data together with the ability of pRB to restrict growth when overexpressed in certain cell types indicate that it functions as tumor suppressor, at least in part, by negatively regulating cell division. Identi®cation of two members of the retinoblastoma family of proteins, p107 (Ewen et al., 1991; Zhu et al., 1993) and p130 (Hannon et al., 1993; Li et al., 1993; Mayol et al., 1993), which share structural and functional similarity with pRB, led to numerous studies seeking to understand whether these two proteins where also tumor suppressors. However, mutations on these genes are rare, and their role as tumor suppressor has not been demonstrated. As pRB, both p107 and p130 are
*Correspondence: X GranÄa, Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, AHP bldg., room 553, 3307 North Broad St., Philadelphia, PA 19140, USA
capable of inhibiting growth in certain cell types, and this inhibition depends on their ability to associate with a number of cellular proteins. In general terms, many of the targets of pocket proteins are either transcription factors or proteins involved in the regulation of transcription (reviewed in White, 1997; Johnson and Schneider-Broussard, 1998; Mayol and GranÄa, 1998; Yee et al., 1998). For instance, pRB has been shown to inhibit RNA transcription by the three cellular RNA polymerases. One prominent function shared by the three pocket proteins is the negative regulation of a family of transcription factors collectively designated as E2F (Johnson and Schneider-Broussard, 1998). E2F family members regulate the transcription of several genes, whose products are required either for the regulation of G1 and S phases of the cell cycle or for DNA metabolism. Thus, by negatively regulating E2F, pocket proteins seem to control, at least in part, the progression through the cell cycle and the cell cycle entry/exit transitions. Moreover, pocket proteins have also been implicated in the process of cell differentiation in various cell types. Dierential phosphorylation of pocket proteins The three members of the pRB family are phosphoproteins and their phosphorylation status is modulated during the cell cycle as well as at the cell cycle entry and exit transitions in mammalian cells (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Mihara et al., 1989; Beijersbergen et al., 1995; Mayol et al., 1995; Xiao et al., 1996). Multiple sites are phosphorylated in pRB and most likely in p107 and p130. All sites identi®ed thus far fall within the consensus site of phosphorylation by Cyclin-DepenKinases (CDKs), which are primarily serine/threonine, proline-directed sites. Most of the work done to date has focussed on pRB and most of its in vivo phosphorylation sites are known. However, less is known about which kinases phosphorylate each one of these sites at a particular time, although a large body of evidence indicates that cell cycle-dependent phosphorylation of pocket proteins is catalyzed by a subset of cyclin/CDK holoenzymes that are sequentially activated during the cell cycle (reviewed in Bartek et al., 1996; Mayol and GranÄa, 1997, 1998). Phosphorylation of pocket proteins in mid-to-late G1 phase is functionally important because it is associated with the loss of the growth suppressive activities of the three pocket proteins. The growth suppressive activity of pocket proteins is mediated through the association
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with a number of cellular proteins, among them, the members of the E2F family of transcription factors, which regulate the expression of a number of genes required for cell cycle progression. Cyclin/CDK complexes collaborate to phosphorylate pocket proteins Progression through mid G1 and S phases from a quiescent stage or from a previous mitotic phase is associated with the sequential activation of a subset of cyclin-dependent kinases (reviewed in GranÄa and Reddy, 1995). D-type cyclin/CDK complexes are activated upon growth factor stimulation of quiescent cells and are kept active through G1. Cyclin E/CDK2 kinase activity increases from mid-to-late G1. Activation of these two subtypes of cyclin/CDK complexes correlates with hyperphosphorylation of the three pocket proteins from mid-to-late G1. Cyclin A/CDK2 complexes, whose activities peak in S phase, are also likely to contribute to the maintenance of the phosphorylation pool of pocket proteins. This hyperphosphorylated state is kept through the remaining of the cell cycle to mitosis, where these forms shift to a hypophosphorylated state by the action, at least in the case of pRB, of a protein phosphatase (reviewed by Ludlow and Nelson, 1995). All these cyclin/CDK complexes are capable of phosphorylating each pocket protein in vitro. In the case of pRB (this has not been addressed for p107 and p130), phosphorylation in vitro occurs at sites known to be phosphorylated in vivo (Lees et al., 1991; Lin et al., 1991; Kato et al., 1993; Kitagawa et al., 1996; Knudsen and Wang, 1996; Connell-Crowley et al., 1997; Zarkowska and Mittnacht, 1997; Zarkowska et al., 1997), and abrogates pRB interaction with E2F complexes (Suzuki-Takahashi et al., 1995; Zhu et al., 1995b). Similarly, isolated native p130/E2F-4 complexes are also disrupted by cyclin/CDK complexes in vitro (Mayol et al., 1996). In agreement with the in vitro experiments, ectopic overexpression of cyclins D1, E or A together with pRB lead to hyperphosphorylation of pRB and abrogates pRB growth inhibitory activity in SAOS-2 cells, which do not express a functional pRB (Hinds et al., 1992; Horton et al., 1995). It is likely, however, that in vivo abrogation of pocket protein/E2F complexes requires the sequential action of D-type cyclins and cyclin E (Lundberg and Weinberg, 1998). Recent studies have revealed dierences in the phosphorylation of speci®c sites on pRB by the above mentioned G1/S cyclin dependent kinases (Kitagawa et al., 1996; Connell-Crowley et al., 1997; Zarkowska and Mittnacht, 1997; Zarkowska et al., 1997). However, these results are controversial since there are reports that do not seem to agree in the phosphorylation speci®city of certain sites by a particular cyclin/CDK complex. A particular residue in pRB, Ser-795, which is eciently phosphorylated by CDK4/cyclin D1 complexes, is not phosphorylated by either CDK2/ or CDK3/cyclin complexes in vitro (Connell-Crowley et al., 1997). CDK2 or CDK3 phosphorylate multiple other sites resulting in hyperphosphorylated pRB, and this action results in the abrogation of the pRB/E2F interaction in vitro. However, this in vitro hyperphosphorylated pRB form is still capable of suppressing growth when microinjected in SAOS-2 cells, suggesting
that phosphorylation of Ser-795 by cyclin D1/CDK-4 complexes is necessary for inactivation of the growth suppressor activity of pRB in SAOS-2 cells (ConnellCrowley et al., 1997). These results highlight the importance of the phosphorylation of particular sites versus the idea that extensive phosphorylation of pRB is required for its inactivation, and suggest that cyclin D-associated kinase activities are essential for the functional inactivation of pRB. A dierential role for cyclin D1 and cyclin E in pRB phosphorylation was suggested by experiments conditionally expressing these cyclins in serum starved and re-stimulated Rat-1 cells. Although premature ectopic expression of both cyclins reduced the length of the G1 phase, only cyclin D1 expression led to premature hyperphosphorylation of pRB (Resnitzky et al., 1994; Resnitzky and Reed, 1995). A dierent experimental approach was based on selectively blocking the activity of D type cyclin/CDK and cyclin E/CDK2 complexes using the CDK inhibitor p16 and a dominant negative CDK2 mutant (dnCDK2) respectively (Lundberg and Weinberg, 1998). These experiments suggested that endogenous D-type cyclins phosphorylate pRB only partially and that cyclin E/CDK2 complexes cannot phosphorylate pRB without previous phosphorylation by D-type cyclin/CDK complexes. Interestingly, phosphorylation of pRB in vivo by presumably endogenous D-type cyclin/CDK complexes in the presence of dnCDK2, which block cyclin E/CDK2 activity, is not sucient to abrogate binding to GSTE2F in vitro. This is in agreement with the ability of dnCDK2 to repress E2F-dependent transactivation (Lundberg and Weinberg, 1998). Phosphorylation of speci®c sites on pRB has also been linked to the dierential abrogation of the interaction between pRB and a set of pRB associated proteins. Phosphorylation of serines 807 and 811 is required to abrogate the interaction between pRB and c-Abl, while phosphorylation of threonines 821 and 826 is required to abolish pRB binding to proteins containing LXCXE motifs such as the SV40 large T antigen (Knudsen and Wang, 1996). In this regard, threonines 821 and 826 are phosphorylated in vitro by cyclin A/CDK2 and cyclin D1/CDK4 respectively. Phosphorylation by either complex will preclude pRB binding to T antigen, however only cyclin A/CDK2 complexes can disrupt preformed complexes. This seems to be due to the inability of cyclin D1/CDK4 to phosphorylate threonine 826 if pRB is pre-bound to a LXCXE containing protein (Zarkowska and Mittnacht, 1997). The apparent contradictory conclusions in some of the above mentioned studies are certainly due to a diculty of studying phosphorylation of pRB in its physiological cellular context. However, a tentative model can be drawn (Figure 1). It seems likely that each G1/S cyclin/CDK complex phosphorylates pRB and related family members at particular sets of phosphorylation sites and at speci®c times during progression through the cell cycle. The nature of the proteins associated to a particular pocket protein, or the absence of these proteins is very likely to aect the ability of cyclin/CDK complexes to phosphorylate particular sites. In addition, previous phosphorylation by a cyclin/CDK complex, might result in conformational changes on the pocket protein that convert it
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Figure 1 Dierential 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 we will discuss later, cyclin/CDK complexes form stable complexes with pocket proteins. D-type cyclins interact with pocket proteins through their LXCXE motif (Dowdy et al., 1993; Ewen et al., 1993; Kato et al., 1993), while cyclin E and cyclin A/CDK2 complexes form stable complexes with p107 and p130, but do not with pRB. Thus, it is conceivable that the activity of a particular cyclin/CDK is necessary to promote and/or negate the action of a dierent cyclin/CDK complex. Phosphorylation of particular sites, or sets of sites, is likely to result in the sequential disruption of pocket protein complexes containing dierent transcription factors leading to the orchestrated succession of events required to promote cell growth and progression through the cell cycle. Is pRB the only essential substrate of D-type cyclin/ CDK complexes? Although all three known pocket proteins are likely to be phosphorylated by D-type cyclin/CDK complexes, it has been suggested that pRB is the only essential
substrate for these CDKs. This conclusion is based on experiments that demonstrate that the essential function of D-type cyclins in G1 depends upon the expression of a functional pRB. In these experiments, microinjected cyclin D1 anti-sense cDNAs or anti-cyclin D1 antibodies were shown to block G1 progression only in cells containing functional pRB (Lukas et al., 1994, 1995a; Tam et al., 1994). In agreement with this notion, the CKI p16 only blocks G1 progression in cells with functional pRB (Lukas et al., 1995b; Medema et al., 1995). Hatekayama and collaborators however, have recently shown that wild type pRB and a phosphorylation resistant pRB mutant do not block cell cycle progression in at least two dierent mouse cytokinedependent hematopoietic cell lines (a pro-B cell line and myeloid 32Dcl3 cells) when ectopically expressed to supra-physiological levels (Hoshikawa et al., 1998). Instead, p130 eciently blocked cell cycle progression through its E2F-binding domain in the mouse pro-B cell line. In these cells, p130 associates with E2F-4 and ectopic overexpression of E2F-4 was sucient to render the cells cytokine-independent (Hoshikawa et al., 1998). It would be interesting to learn whether p130 is an
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essential target for D-type cyclin/CDK holoenzymes in these cells. Consistent with this, p130 and p107 containing complexes, but not pRB complexes, are the major components of E2F-DNA-binding activities in mouse T lymphocytes progressing through the cell cycle (Cobrinik et al., 1993). In normal mouse T lymphocytes, however, ablation of p130 or p130/p107 functions, is compensated by p107 or pRB, respectively (Mulligan et al., 1998). These experiments suggest that although pRB is a critical cell cycle regulator in some cell types, p130 function is also critical in certain cell types such as hematopoietic cells. Dierential regulation of pocket proteins by phosphorylation p107 and p130 exhibit a simpler pattern of dierently phosphorylated forms than pRB when resolved by SDS ± PAGE followed by Western blot analysis (Figure 1). Several pRB forms with dierent mobility are detected during a complete cell cycle, and the patterns of pRB forms change signi®cantly from cell type to cell type. In contrast, two dierent p107 forms are detected in most cell types and these forms are generally designated as hypo- and hyperphosphorylated p107. In the case of p130, four forms are detected in most cell types, which are designated unphosphorylated p130 and phosphorylated p130 forms 1, 2 and 3 (Mayol et al., 1995). Interestingly, each one of these forms can be associated with a particular stage of the cell cycle or cell quiescence (Mayol et al., 1996; Garriga et al., 1998). Particular attention needs to be taken to the antibodies used to detect phosphorylated forms of pocket proteins in immunoprecipitation and Western blot experiments. It is well known that some of the available antibodies (especially some monoclonal antibodies), often recognize only a subset of forms. As we have discussed above, phosphorylation of the three pocket proteins during the cell cycle is likely to occur through shared cyclin/CDK-dependent pathways. Activation of cyclin/CDK complexes in mid-tolate G1 results in the hyper-phosphorylation of pRB and p107 and in the phosphorylation of p130 to form 3. In contrast, at the G1/G0 transition, when cells exit the cell cycle to a resting state, p130 phosphorylation status is modulated dierently from pRB and p107 (Mayol et al., 1996; Garriga et al., 1998). While pRB and p107 become hypophosphorylated, p130 is detected as phosphorylated forms 1 and 2. This unique modulation of p130 phosphorylation status correlates with a dramatic accumulation of p130 protein and p130/E2F complexes, which are characteristic of a quiescent stage. Because this phosphorylation event that speci®cally targets p130 is maintained in quiescent cells and readily detected in adult tissues, when no G1, S and G2/M cyclin/CDK activities are detectable, we postulated that a dierent kinase activity targets p130 at the cell cycle exit and during the quiescent stage (Mayol et al., 1995, 1996; Garriga et al., 1998). It is still unclear whether this kinase is a dierently regulated CDK family member, or conversely, another unrelated kinase (reviewed in Mayol and GranÄa, 1998). A transient block in G1 is induced by TGF-b in exponentially growing HaCaT cells. This block in G1
results in the primary accumulation of p130 form 1 and depletion of p130 form 3, correlating with a TGF-bmediated inactivation of G1 cyclin/CDK complexes. This transient block in G1 should not be confused with a G0 quiescent stage, in which p130 from 2 is the primary form of p130 in these cells (Mayol et al., 1996). In agreement with these results, p130 shift to form 3 is blocked in serum starved and re-stimulated HaCaT cells progressing through early G1 in the presence of TGF-b. However, p130 form 2 does not accumulate as a consequence, suggesting that p130 form 2 is not just an intermediate stage of phosphorylation due to lack of activation of G1 cyclin/CDK complexes (Mayol et al., 1996). In agreement with the changes in p130 phosphorylation status described above, cell cycle exit to G0 by serum starvation of HaCaT cells results in the accumulation of p130/E2F-4 complexes, while a TGF-b-mediated G1 arrest does not (Belbrahem et al., 1996). Modulation of pocket protein levels Although the levels of pRB are quite constant during the cell cycle and in quiescent cells, levels of p130 and p107 change dramatically depending upon the stage of the cell cycle (Beijersbergen et al., 1995; Mayol et al., 1995, 1996). p130 is detected in quiescent cells as two abundant phosphorylated forms (forms 1 and 2). When cells progress through the G0/G1 transition and through mid G1, p130 is hyperphosphorylated to form 3, whose levels are dramatically downregulated as cells move to S phase (Mayol et al., 1995, 1996). In contrast, when cycling cells are forced to exit the cell cycle, p130 protein levels raise sharply (p130 forms 1 and 2) coinciding with the accumulation of p130/E2F complexes (Kiess et al., 1995; Mayol et al., 1996; Smith et al., 1996; Garriga et al., 1998). The increase of p130 levels and p130/E2F protein complexes in response to cell cycle exit signals occurs from a period during the G1 phase previous to the restriction point, where otherwise p130 would be phosphorylated to form 3 (Mayol et al., 1996). This was proved in experiments where synchronous populations of cells at particular stages of the cell cycle were forced to exit the cell cycle. Placing post-restriction point cells, which have not reached mitosis, in environmental conditions leading to cell cycle exit did not result in accumulation of p130 or in the accumulation of p130/ E2F complexes until the post-mitotic G1 phase. These experiments linked p130 accumulation to the G1/G0 transition and not to any transient cell cycle arrest at other cell cycle points (Mayol et al., 1996). Since the decision to exit the cell cycle does not occur until the post-mitotic early G1 phase, accumulation of p130 might regulate the decision to exit the cell cycle. Accumulation of hypophosphorylated pRB in these conditions is likely to play a role in the shutting-o of E2F-dependent transcription (Ikeda et al., 1996). Whether pRB and p130 act individually or a concerted action of both proteins regulates this step is not yet clear, and it might be cell type-dependent (Hoshikawa et al., 1998). The study of mice harboring mutations in genes encoding pocket proteins has provided some clues. However, the compensatory ability of the remaining pocket protein(s) to partially
The pRB family in negative cell growth control X GranÄa et al
substitute for the absent one has made these studies dicult to interpret. The levels of p107 are modulated in an opposing manner to p130. Very little-to-non p107 is detected in quiescent cells. However, p107 accumulates sharply as cells progress through the cell cycle (Beijersbergen et al., 1995). The increase in p107 protein levels correlate with an increase in p107 mRNA transcripts, indicating that p107 protein expression is regulated at the transcriptional level (Hurford et al., 1997). Interestingly, p107 is under negative control mediated through E2F sites on its promoter, suggesting that either pRB, p130 or both are involved in the control of p107 expression (Zhu et al., 1995c). The existence of connecting regulatory loops among pocket proteins ensures that their levels oscillate coordinately during the cell cycle and at the G0/G1 and G1/G0 transitions in a way that allows an orchestrated regulation of common target factors (reviewed in Mayol and GranÄa, 1998). On the other hand, very little oscillation of the levels of p130 mRNA transcripts is observed in quiescent and cycling cells (Richon et al., 1997; Smith et al., 1998), indicating that changes in protein levels are primarily post-transcriptional. Thus, p130 levels are regulated by the rate of protein synthesis and/or by changes in protein stability linked to speci®c cell cycle stages. In this regard, there is a striking correlation between the phosphorylation status of p130 and its protein levels, suggesting that phosphorylation of speci®c sites regulates the stability of the p130 protein. The phosphorylation events leading to the generation of p130 form 3 in mid G1 are followed by dramatic downregulation of p130 (Mayol et al., 1995, 1996), but the mechanisms mediating this downregulation are still unclear. Since
a number of cell cycle regulators, including p27, cyclin D1 and cyclin E, are targeted by the proteasome machinery upon phosphorylation of particular CDK consensus sites (Clurman et al., 1996; Won and Reed, 1996; Diehl et al., 1997; Shea et al., 1997), it is possible that p130 downregulation is mediated by a cell cycle stage-dependent proteasome degradation pathway. In support of increased degradation of p130 during S phase, hyperphosphorylated p130 form 3 is metabolically labeled more eciently than G0phase p130 forms, thus suggesting that the rates of synthesis and phosphorylation are higher in S phase and, therefore, G0-phase p130 forms must be more stable than p130 form 3 (Kiess et al., 1995; Mayol et al., 1995). Moreover, preliminary experiments using proteasome inhibitors block p130 depletion in S phase, but cell cycle progression through S phase in the analysed cells was also delayed by these inhibitors and the involvement of the proteasome complex may be indirect (Smith et al., 1998). In this context, we have found that cellular treatments that delay the progression of the cell cycle through S phase, even a slight eect, also delay the downregulation of p130 (unpublished observations). Thus, although the above cited hypothesis is an attractive possibility, the mechanism of p130 downregulation is yet to be clearly demonstrated. Finally, at the cell cycle exit, dramatic accumulation of p130 occurs without changes in p130 mRNA levels, suggesting that p130 accumulation is regulated at a post-transcriptional level. Consistent with this, cell cycle exit phosphorylation of p130 indeed results in stabilization of the p130 protein since p130 form 2 accumulates while the metabolic labeling rates of p130 are reduced during quiescence (Mayol et al., 1995, 1996; Garriga et al., 1998).
Table 1 Cellular protein targets of the pRB family of proteins (see text for references) Associated protein
Biochemical function
Pocket protein
Biological role
Cyclin D Cyclins A and E RbK E2F family c-Jun ATF-2 c-Myc, N-Myc Spl Abl MDM2 MCM7 HNuc RBAp48 AhR TAFII250/TFIID TFIIIB UBF HDAC1 MyoD HBP1 p202 C/EBP, NF-IL6 NRP/B PLH proteins BRG1 hBRM Id-2 AP-2 Trip230
CDK subunit CDK subunits Protein kinase Transcription factors Transcription factor Transcription factor Transcription factors Transcription factor Nuclear tyrosine kinase Nuclear protein DNA replication licensing Nuclear protein Transcription factor-corepressor Transcription factor Transcription factor RNA pol III-Transcription factor RNA pol I-Transcription factor Histone deacetylase Transcription factor Transcription factor Transcription factor Transcription factor Nuclear matrix Transcription factor Transcription factor Transcription factor-coactivator Transcription factor-corepressor Transcription factor THR-coactivator
pRB p107, p130 pRB pRB, p107, p130 pRB pRB pRB, p107 pRB, p107 pRB pRB pRB, p107, p130 pRB pRB pRB pRB pRB pRB pRB, p107, p130 pRB, p107 pRB, p130 pRB pRB pRB pRB, p107, p130 pRB pRB pRB pRB pRB
Cell cycle Cell cycle Cell cycle? Cell cycle Cell cycle? TGF-beta expression Cell cycle? Cell cycle? Cell cycle? pRB inhibitor? DNA replication inhibition? Mitosis? Growth inhibition? TCDD-growth arrest Transcription tRNA synthesis rRNA synthesis Transcription Muscle dierentiation Muscle dierentiation Muscle dierentiation Adipocyte dierentiation Neuronal dierentiation Homeosis? ? Glucocorticoid response? Cell cycle? Epithelial dierentiation? Thyroid hormone response
3369
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Biochemical consequences of the regulation of pocket proteins As discussed later, the biochemical action of pocket proteins is largely dependent on their ability to associate and thereby modulate the activities of cellular proteins (Table 1). These associations involve a wide range of transcription factors, as well as enzymes such as histone deacetylase, through which pocket proteins are thought to regulate gene expression. Indeed, through interactions with various transcription factors, pRB family members are capable of regulating the activity of the three RNA polymerases (reviewed by Dynlacht, 1995; White, 1997). Pocket proteins also associate with tissue speci®c transcription factors including MyoD, HBP1, C/EBP, and NRP/B, among others, which may be important mediators of the biological role of pocket proteins in cell dierentiation processes. Moreover, pocket proteins associate with dierent cyclin/CDK complexes, perhaps, modulating their activity, and recent ®ndings suggest that they may also interact directly with components of the DNA replication machinery. Thus, pocket proteins appear to act at diverse cellular levels to control cell growth and dierentiation processes. Association with cellular proteins The association of pocket proteins with their cellular partners is dependent, in many cases, on the phosphorylation status of the pocket protein. Only unphosphorylated or hypophosphorylated pocket protein forms are able to associate with most of their targets. The inference that lack of cyclin-CDK phosphorylation on pocket proteins is determinant in allowing such protein associations is based on results of experiments in which hyperphosphorylation of pocket proteins correlated with protein complex disruption. Conversely, pocket protein hypophosphorylation correlates with the appearance of such complexes. In support of this, in vitro experiments have shown that CDK activities directly disrupt or impede pocket protein/E2F complexes (Suzuki-Takahashi et al., 1995; Zhu et al., 1995b; Mayol et al., 1996). The kinetics of activation/inactivation in vivo of the cyclin/CDK complexes used in these in vitro experiments correlates with the status of pocket protein phosphorylation observed during cell cycle progression and growth arrest. Although direct proof for this phosphorylation-dependent association with cellular proteins has not been assessed in all the instances, this mechanism is assumed to be a general mechanism governing interactions between these proteins (reviewed in Mayol and GranÄa, 1997, 1998; Johnson and Schneider-Broussard, 1998; Yee et al., 1998). In some cases, association of pocket proteins with certain cellular proteins may apparently be without the restrain imposed by CDK phosphorylation in G1. These cases include the association with certain cyclin/ CDK complexes by direct binding, which occurs during cell cycle phases when the activities of these kinases are high and pocket proteins are primarily hyperphosphorylated (Figure 1). In particular, hyperphosphorylated p130 possesses a transient histone H1-associated kinase activity during the cell cycle, most likely
composed of CDK2, which is most active in late S and G2 phases (Mayol et al., 1995; Vairo et al., 1995). Similarly, p107 associates with cyclin E/CDK2 and cyclin A/CDK2 complexes in late G1 and during the S phase (Lees et al., 1992; Shirodkar et al., 1992; Chittenden et al., 1993; Cobrinik et al., 1993; Schwarz et al., 1993). These data are in support of an active role of pocket proteins during stages when they are thought to be inactivated by CDK-mediated hyperphosphorylation. As discussed earlier, multiple amino acid sites in pocket proteins are coordinately phosphorylated by diverse CDKs, and suppression of binding abilities to particular cellular proteins may thus depend on the status of key phosphorylation sites. Moreover, although direct evidence for the in vivo binding of hyperphosphorylated pocket protein forms to transcription factors or to other cellular proteins has not been reported, hyperphosphorylation of p107 and p130 does not aect their binding ability to the SV40 large T antigen-a LXCXE-containing viral protein. In contrast, binding of pRB to this viral protein is largely dependent on the phosphorylation of threonine 821 not conserved in p130 or p107 (Knudsen and Wang, 1998). These observations indicate that cyclin-CDK activation in G1 may not result in the absolute disruption of pocket protein associations and that hyperphosphorylated pocket protein forms still retain the ability to form certain protein complexes. Pocket protein association with cellular proteins is also regulated by modulation of the pocket protein levels in the cell. This is particularly important in the case of p130 and p107, which display a high degree of biochemical similarity and show opposite patterns of expression in diverse instances of cell growth and dierentiation (reviewed in Mayol and GranÄa, 1997, 1998). Finally, availability of cellular targets to pocket proteins is a determinant in the modulation of cellular pathways that depend on pocket protein function. Thus, in processes such as cell dierentiation that require pocket protein-mediated regulation of gene expression, tissue-speci®c transcription factors need to be expressed and exposed to pocket proteins in order to be biochemically activated or inactivated. Transcriptional repression mediated by E2F The E2F family of transcription factors is composed of six members (E2F-1 to -6) that form DNA-binding heterodimers with members of the related DP family of co-activators (DP-1 to DP-3). In proliferating cells, E2F proteins activate transcription of several genes important for cell cycle progression (for recent reviews see Johnson and Schneider-Broussard, 1998; Mayol and GranÄa, 1998, and references therein). In contrast, in growth-inhibited cells as well as at initial stages of G1 progression, E2F proteins are targeted by pocket proteins and are converted into transcriptional repressors of many genes that are positively regulated during the cell cycle. The recently identi®ed E2F-6, diers from other members of the family in that it does not posses a pocket protein binding domain, yet, seems to act as a repressor of E2F-site-dependent transcription (Morkel et al., 1997; Cartwright et al., 1998; Gaubatz et al., 1998; Trimarchi et al., 1998). Pocket proteins associate dierentially with certain E2F family
The pRB family in negative cell growth control X GranÄa et al
members both in vivo and in vitro, p130 and p107 exclusively targeting E2F-4 and E2F-5, and pRB targeting E2F-1, E2F-2, E2F-3 and E2F-4 (Figure 2; reviewed in Johnson and Schneider-Broussard, 1998; Mayol and GranÄa, 1998). The genes transcriptionally regulated by E2F transcription factors include genes coding for cell cycle regulatory proteins: cyclin E (Ohtani et al., 1995; Botz et al., 1996; Geng et al., 1996), cyclin A (Schulze et al., 1995; Zerfass et al., 1995; Huet et al., 1996; Philips et al., 1998), CDC2 (Dalton, 1992; Tommasi and Pfeifer, 1995), CDC25C (Zwicker et al., 1995a, b), the CDK inhibitor p21 (Hiyama et al., 1998) and protooncogenes such as Myc and Myb (reviewed in Slansky and Farnham, 1996). Interestingly, p107 (Zhu et al., 1995c), E2F-1 (Hsiao et al., 1994; Johnson et al., 1994; Neuman et al., 1994; Johnson, 1995; Smith et al., 1996), and E2F-2 genes (Smith et al., 1996; Sears et al., 1997) are also under E2F transcriptional control during the cell cycle, which may have relevant implications in the coordination of individual pocket protein functions and is discussed later in this section. Moreover, proteins involved in DNA metabolism and some constituents of DNA replication complexes are also transcriptionally regulated by E2F proteins: dihydrofolate reductase (DHFR) (Means et al., 1992; Slansky et al., 1993), thymidine kinase (TK) (Dou et al., 1992; Ogris et al., 1993), thymidylate synthase (TS) (Johnson, 1992), histone H2A (Oswald et al., 1996), Orc1 (Ohtani et al., 1996), and CDC6 (Ohtani et al., 1998; Yan et al.,
1998). The role of these E2F-regulated genes in cell growth processes, together with the fact that activation of E2F family members is sucient to induce DNA replication and that pocket protein suppressive function on the cell cycle is largely dependent on the interaction with E2F proteins (reviewed in Johnson and Schneider-Broussard, 1998), indicates that pocket proteins and E2F play a central role in the control of the cell division cycle. Pocket protein phosphorylation by cyclin/CDKs during the G1/S transition disrupts E2F-pocket protein complexes, both, relieving their repressive action on gene expression and releasing active E2F transcription factors. Consequently, activation of E2F in G1 allows the transcription of genes required for cell cycle progression. In this scenario, a number of aerent growth signals, either activating or suppressing growth, are integrated by cyclin-CDKs leading to modulation of the phosphorylation status of pocket proteins, which ultimately determines the relative action of E2F as a transcriptional activator or repressor. Therefore, it is widely accepted that this pathway constitutes one of the main steps in the decision of passage through the G1 restriction (R) point, from which cells are committed to cell cycle progression independently of external growth signals. Conversely, if growth inhibiting signals dominate, this pathway is blocked at particular points and, eventually, cells may chose to exit the cell cycle. Some of the genes whose products are required for 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
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repressed by E2F in non-dividing cells. Since E2F-4 is the main E2F family member found in quiescent cells, and is mostly found in association with p130 in a wide range of quiescence and dierentiation models (reviewed in Johnson and Schneider-Broussard, 1998; Mayol and GranÄa, 1998), it is assumed that E2Fmediated repression of gene expression, upon cell cycle exit, is primarily exerted by p130/E2F-4 complexes. It must be noted that, pRB has also been reported to complex with E2F in some quiescent cells (Corbeil et al., 1995; Halevy et al., 1995; Ikeda et al., 1996; Rampalli et al., 1998). Furthermore, serum-deprived pRB7/7 mouse ®broblasts show deregulated expression of some E2F-dependent genes, which suggests that pRB plays a role similar to p130, at least in the repression of certain genes. One of the mechanisms that may be critical in the formation of these major p130/E2F-4 complexes is the accumulation of p130 protein upon cell cycle exit, probably by protein stabilization, and downregulation of p107, whose major partner is E2F-4 (Kiess et al., 1995; Mayol et al., 1996; Smith et al., 1996, 1998; Garriga et al., 1998; reviewed in Mayol and GranÄa, 1998). The reason why pRB is not found appreciably associated with E2F after cell cycle exit is not clear, but the observed higher anity of E2F-4 for p130 binding in vitro (Sardet et al., 1995) suggests that the accumulation of p130 displaces pRB from associating with E2F-4 complexes. The fact that p1307/7 p1077/7 MEFs and lymphocytes show upregulated expression of some E2F-dependent genes normally repressed in G0 indicates that pRB is unable to substitute for all p130 mediated transcriptional repression events. Conceivably, pRB might also repress the expression of some genes in G0 through interaction with transcription factors other than E2F. The biochemical events during G1 progression must be very dierent depending on whether we consider cells entering the cell cycle from G0 or cells after having exited mitosis in a continuously proliferating state. Particularly, G1 re-entrance from G0 involves an early stage where pre-existing p130/E2F-4 complexes are still abundant and repress transcription from E2Fdependent promoters, whereas cells exiting mitosis into G1 characteristically contain pRB/E2F complexes (Mayol et al., 1996; Smith et al., 1996). These patterns are consistent with a dierential role of pRB in the R point passage and of p130 in G0/G1 transitions (Mayol et al., 1996). Quiescent cells do not express many cell cycle regulators, which must be coordinately resynthesized during G1 for a proper cell cycle reentrance. In this context, persistence of the cell cycle exit-associated p130 phosphorylation that stabilize its protein levels seem to be crucial in keeping E2F-4 as a repressor during early G1. Further progression through the G1 phase leads to cyclin/CDK activation, and concomitant hyperphosphorylation of p130 to form 3 disrupts p130/E2F-4 complexes (Mayol et al., 1996), thereby releasing E2F-dependent transcriptional repression to allow for the expression of genes required for passage through the R point and further cell cycle progression. E2F-1 and E2F-2 are under E2F-dependent transcriptional control during the cell cycle, being not expressed in resting cells and expressed in mid G1 coinciding with cyclin/CDK activation (Hsiao et al., 1994; Johnson et al., 1994; Neuman et al., 1994;
Johnson, 1995; Smith et al., 1996; Sears et al., 1997). It is thus assumed that one of the consequences upon disruption of p130/E2F-4 complexes by G1 cyclin/ CDKs is the transcriptional activation of E2F-1 and E2F-2. Since E2F-1 and E2F-2 speci®cally associate with pRB, but not with p130 or p107, and since pRB rarely associates with E2F-4 in the presence of p130/ E2F-4 complexes, these data again suggest that the pRB-E2F interaction during G1 is largely dependent on previous disruption of p130/E2F-4 complexes. This scenario further implicates pRB as a downstream eector of p130 function during the G0/G1 transition. In contrast, only E2F-3 DNA-binding activities re-accumulate in post-mitotic G1 Ref 52 cells (Leone et al., 1998). Most importantly, antibodies raised to E2F3, but not anti-E2F-1 antibodies, microinjected in Ref 52 cells synchronized in G2 resulted in inhibition of S phase-entry of the next cell cycle. Therefore, the mechanisms that regulate the expression of E2Fdependent genes are dierent in cells entering the cell cycle from a quiescent stage or in proliferating cells. The p107 gene is also silent in resting cells at least partially due to E2F-dependent transcriptional repression, and is re-activated in mid G1 during cell cycle progression (Beijersbergen et al., 1995; Kiess et al., 1995; Hurford et al., 1997; Garriga et al., 1998). These observations in conjunction with the opposing patterns of expression of p130 and p107 protein levels in a number of cell growth and dierentiation models (Kiess et al., 1995; Richon et al., 1997; Garriga et al., 1998) strongly suggest that p107 is under the control of p130, possibly through E2F-dependent transcriptional regulation. However, serum starved p1307/7 mouse ®broblasts show abnormally elevated levels of p107 protein, but, p107 mRNA remains unchanged, suggesting that the eect of p130 on p107 expression is posttranscriptional at least in this cell type (Hurford et al., 1997). In contrast, serum-starved pRB7/7 ®broblasts do show elevation of p107 mRNA levels which suggests that pRB is a better candidate than p130 in conferring the E2F-dependent transcriptional repression on p107 at least in these cells. Regarding other E2F-taget genes known to be upregulated during G1, it is dicult to distinguish whether they are directly activated by disruption of p130/E2F-4 complexes or if subsequent expression of pRB-associated E2F members and activation of pathways downstream of p130 are also involved. One should keep in mind that although the above mentioned genes are thought to be repressed through E2F sites in G0, their expression timing during the cell cycle re-entry diers signi®cantly. In the case of cyclin A, for instance, it seems that its E2F dependent expression in late G1 results from the sequential repressive action of the three pocket proteins during G1 progression. Recent experimental evidence indicate that the repressive action of E2F-pocket protein complexes on cyclin gene expression is tightly regulated during G1 and it plays a major role in the R point transition. The G1 arrest mediated by a pRB phosphorylation mutant constitutively active in the binding to E2F allows the expression of cyclins D and E, but not cyclin A (Knudsen et al., 1998). Since p130 and p107 display hyperphosphorylation under this G1 arrest condition, these results suggest that the G1 phase has progressed to a pRB-sensitive critical point downstream of cyclin
The pRB family in negative cell growth control X GranÄa et al
D and E expression and activation of their associated kinase activity. The fact that cyclin E expression is unaected by the pRB phosphorylation mutant suggests that E2F-dependent transcriptional activation of the cyclin E gene during G1 takes place after phosphorylation of p130. Importantly, increasing the levels of cyclin E in these cells bypasses the G1 arrest imposed by the pRB mutant, or by the CDK inhibitor p16 (Alevizopoulos et al., 1997; Lukas et al., 1997), and allows S phase entrance, suggesting that overexpression of cyclin E can activate pathways of passage through the R point independent or downstream of pRB/E2F control. With respect to cyclin A, its timing of expression beginning in late G1 and its dependence on cyclin E activation (Zerfass-Thome et al., 1997) suggest that cyclin A expression is secondary to the R point passage. In this context, it has been suggested that multimeric complexes containing p107/E2F and cyclin E/CDK2 are targeted to the cyclin A gene promoter and that CDK2 activity is required for cyclin A promoter activation in late G1 (Zerfass-Thome et al., 1997; Schulze et al., 1998). Ectopic expression of cyclin E allowed for the expression of cyclin A in the presence of the pRB phosphorylation mutant (active pRB), but the cells arrested during S phase progression (Knudsen et al., 1998). Thus, these results suggest that phosphorylation of pRB after the G1/S transition is required. Keeping pRB, and perhaps other pocket proteins, in its hyperphosphorylated state should potentially allow for the continued expression of certain genes required in S phase. Finally, recent experiments suggest that, once postrestriction point commitment has taken place, pocket proteins exert a direct role in modulating DNA replication during S phase. As seen earlier, overexpression of cyclin E bypasses a G1 arrest induced by a constitutively active pRB mutant and results in the entrance into S phase. However, cells do not complete S phase, which reveals an inhibitory function of pRB on DNA replication (Knudsen et al., 1998). In this respect, modulation of pRB/E2F and p107/E2F complex levels during S phase might have a role in controlling the expression of certain genes required for DNA replication. Moreover, a G2 phase block induced by the CDK inhibitor p21 is accompanied by DNA endo-reduplication in the absence of functional pRB suggesting that suppression of DNA synthesis after S phase completion is, at least partially, controlled by pRB in G2 arrested cells (Niculescu et al., 1998). Interestingly, direct binding of pRB and p130 to MCM7 protein, a DNA replication licensing factor, has recently suggested that pocket proteins may directly regulate the activity of these complexes by direct protein-protein associations (Sterner et al., 1998). Further studies, however, are needed to ascertain the functional extent of pocket protein interactions with the DNA replication machinery. Histone deacetylase As discussed earlier, pocket proteins actively repress the transcription of E2F dependent genes. This occurs, at least in part, because of the ability of dierent E2F/ DP complexes to tether pocket proteins to the E2F sites contained in responsive promoters. Repression appears to be due to the ability of pRB (Brehm et al.,
1998; Ferreira et al., 1998; Luo et al., 1998; MagnaghiJaulin et al., 1998) and the other two family members, p107 and p130 (Ferreira et al., 1998) to recruit histone deacetylase 1 (HDAC-1) to E2F site-containing promoters (Figure 3a). Histone deacetylases are enzymes that mediate the removal of acetyl groups from core histones and other proteins involved in transcription. This causes reorganization of chromatin that impairs the access of transcription factors to DNA and thereby inhibits transcription. Recently it has been shown that a deacetylase activity associates with pRB both in vitro and in vivo. This associated activity is catalyzed by the HDAC-1 protein, which associates with pRB. pRB interacts, through its A/B pocket domain, with an LXCXE-like motif (IACEE) located in the C-terminus of HDAC-1. The C-terminus of pRB is dispensable for such interaction (Magnaghi-Jaulin et al., 1998), which is in agreement with the fact that the A/B pocket is both necessary and sucient for the transcriptional repression activity of pRB (Johnson and SchneiderBroussard, 1998). Moreover, the pRB/HDAC-1 binding can be competed by peptides containing the LXCXE motif of viral proteins known to interact with pRB, suggesting that those viral proteins may release the pRB transcriptional repression by disrupting pRB/HDAC-1 complexes (Brehm et al., 1998; Magnaghi-Jaulin et al., 1998). The notion that repression of genes containing E2F sites by pRB is mediated by HDAC-1 is based on the following pieces of evidence: (i) E2F is not able to associate with HDAC-1 in the absence of pRB, suggesting that pRB tethers HDAC1 to E2F; (ii) in transient cotransfection experiments using a luciferase reporter gene under the control of an E2F promoter and expression vectors for E2F, pRB and HDAC-1, expression of HDAC-1 increased E2F-mediated repression by pRB, whereas in the absence of pRB, HDAC-1 has no eect on the E2F promoter and (iii) this inhibitory eect was also demonstrated using a stably integrated reporter gene (Brehm et al., 1998), which best resembles the real architecture of a gene promoter in its chromosomal context. Since HDAC-1 enhances repression by pRB in both a genomic environment and in non-integrated plasmids, it seems that deacetylation events may also occur in non-chromatin proteins. Although these data indicate that pRB uses deacetylase activity to repress E2F, TSA (a deacetylase inhibitor) does not completely release repression, suggesting that pRB, and by extension other pocket proteins, use additional mechanisms to repress E2F-dependent promoters. Both, p107 and p130, have recently been demonstrated to interact with and repress E2F-4 activity through a histone deacetylase. Thus, it seems that p107 and p130 also tether HDAC-1 or a related deacetylase to E2F promoters, indicating that the three pocket proteins share a similar model of E2F-mediated repression by recruiting HDAC-1 (Ferreira et al., 1998). Transcriptional repression of RNA polymerases I and III by pRB pRB inhibits transcription by both RNA pol I and RNA pol III. RNA pol I is responsible for the transcription of the large ribosomal subunit (rRNA), while RNA pol III is involved in the transcription of
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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 limiting the production of rRNA and tRNA, pRB might repress the level of protein synthesis, which in turn would result in suppression of cell growth (reviewed by White, 1997). TPA-mediated dierentiation of human monocytelike U937 cells correlates with downregulation of rRNA synthesis and results in translocation of pRB to the nucleolus. Since the nucleolus is the site of RNA pol I transcription, these data suggest that pRB could mediate inhibition of transcription of rRNA genes by RNA pol I (Cavanaugh et al., 1995). In support of this hypothesis, pRB has been found to associate with UBF (Upstream Binding Factor), a transcription factor that, although not required for basal rDNA transcription, stimulates RNA pol I transcription by helping assembly of the initiation complex (Figure 3b). In this regard, it has been shown that UBF/pRB interaction increases during U937 dierentiation (Cavanaugh et al., 1995). Moreover, repression of UBF-stimulated RNA pol I transcription requires the C-terminus domain of pRB, being the N-terminus dispensable. Interestingly, mutations on the A/B pocket do not seem to aect the ability of pRB of interacting with UBF. As a result of pRB association with UBF, the binding of
UBF to the rDNA promoter is impaired (Voit et al., 1997). On the other hand, several lines of evidence suggested that pRB also inhibits RNA pol III. The synthesis of RNA pol III products is inhibited when pRB is overexpressed in transfected cells or added in a reconstructed in vitro system. When comparing two osteosarcoma cell lines, which dier in their pRB status, the activity of RNA pol III is much higher in pRB de®cient SAOS-2 cells than in pRB wild type U-2 OS cells. More convincing evidence comes from measurement of RNA pol III activity in mouse embryonic ®broblasts (MEFs) obtained from pRB7/7 mouse embryos. In pRB7/7 MEFs, RNA pol III activity is four to ®ve times higher than in control ®broblasts (White et al., 1996). Since pRB is able to inhibit the transcription from three dierent types of RNA pol III promoters, it was postulated that pRB could mediate this inhibition through a general transcription factor. TFIIIB, which directs RNA pol III to the start site, seems to be such factor. TFIIIB and pRB co-purify during multiple diverse chromatographic steps and a physical interaction between pRB and TFIIIB was demonstrated by immunoprecipitation and recombinant-protein pull-down experiments. pRB might disrupt TFIIIB function by mimicking two of its
The pRB family in negative cell growth control X GranÄa et al
subunits, TBP and BRF (Figure 3c), with which it shares some homology (Larminie et al., 1997). Whether p107 and or p130 regulate RNA pol III is not known. The fact that RNA pol III activity is increased in pRB7/7 MEFs (White et al., 1996), demonstrates that neither p107 nor p130 compensate for this function of pRB in its absence. This result indicates that either p107 and p130 do not regulate RNA pol III or that the levels of these proteins are not sucient to repress RNA pol III in the absence of pRB. The implications of the regulation of RNA pol I and III by pRB in cancer are clear from the following facts: (i) the domains of pRB required for growth suppression are also required for inhibition of the two polymerases by pRB and (ii) naturally occurring mutations that render pRB inactive as tumor suppressor also compromise its ability to inhibit the activity of RNA pol I and III (White, 1997). In agreement with these results, the activities of RNA pols I and III are found elevated in murine tumors and are elevated by transforming agents (reviewed by White, 1997) p107 and p130 as CDK inhibitors Two short amino acid motifs have been identi®ed in p107 and p130, which are required for binding of p107 and p130 to cyclin E/CDK2 or cyclin A/CDK2 complexes (Adams et al., 1996; Lacy and Whyte, 1997; Woo et al., 1997; CastanÄo et al., 1998). Similar short amino acid motifs are found in the CKIs (p21, p27, p57) and E2Fs (E2F-1, -2, -3) that bind cyclins E and A. One of these motifs is present in the spacer region of p130 and p107, but is not found in the highly divergent spacer region of pRB (Adams et al., 1996; Lacy and Whyte, 1997). In agreement with this, pRB does not bind to cyclins E and A. The other motif is found at the amino terminus (Castano et al., 1998). Mutations in either one motif are sucient to abolish the interaction of p107 and p130 with these cyclins (Adams et al., 1996; Lacy and Whyte, 1997; Castano et al., 1998). Since the ®rst binding motif identi®ed in the spacer region of p107 and p130 shared identity with proteins that are either CDK substrates or CKIs (Adams et al., 1996), it was suggested that CKIs could function at least in part by blocking the access of substrates to the kinase catalytic subunit (Adams et al., 1996). The notion that p107 and p130 could act as CKIs was proposed based on the fact that the ability of p107 and p130 to suppress growth depends, at least in part, on their ability to inhibit CDK2 activity (Zhu et al., 1995a, b; Deluca et al., 1997; Woo et al., 1997; Castano et al., 1998). In this context, it has been shown that p107 exhibits an inhibitory constant (Ki) towards cyclin E/CDK2 and cyclin A/CDK2 comparable to that of the CKI p21. In agreement with these results, puri®ed recombinant p107/cyclin/CDK2 or p130/ cyclin/CDK2 complexes possess lower kinase activity towards exogenous substrates such as histone H1 than single cyclin/CDK2 complexes (Woo et al., 1997). Since p107 and p130 are likely in vivo substrates of cyclin/CDK2 complexes, the results obtained by dierent groups summarized above have led to distinct interpretations. These include that: p107 and p130 are preferential substrates (Adams et al., 1995), they change the substrate speci®city of CDK2 (Hauser et
al., 1997) or they act as genuine CKIs (Castano et al., 1998). In support of the later, low levels of ectopic p107 in SAOS-2 cells led to a decrease in CDK2 activity, which occurred without changes in the levels of CDK2 and cyclins A and E, apparently ruling out E2F-mediated repression of cyclin A and cyclin E genes. In keeping with this notion, levels of CDK2 activity were found to be increased in p1077/7 and p1077/7 p1307/7 MEFs (Castano et al., 1998). Nevertheless, the possibility that these in vivo eects are indirect has not been ruled out. Although the biochemical grounds supporting a function for p107 and p130 as CKIs are sound, one should keep in mind the relative abundance of these proteins and the complexes that contain them in dierent physiological cellular instances. This is particularly important for p130 since its levels drop dramatically as cells progress through S phase and mitosis. Thus, the relative levels of p130/cyclin (E or A)/CDK2 complexes compared to p107/cyclin (E or A)/CDK2 complexes and/or other cyclin (E or A)/ 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, accumulation of p130 only occurs in the G1 phase before the G1/G0 transition point, at a time when the CDK2 associated cyclin E and cyclin A kinase activities are already low due to normal cell cycle dependent regulation (Mayol et al., 1996). Moreover, we should also consider that a number of cellular pathways, which lead to cell cycle exit, result in accumulation and binding of CKIs to their corresponding CDK or cyclin/CDK targets, but no increase of p107 and/or p130 bound to cyclin/CDK2 complexes has been as to yet reported. Indeed, binding of p21 and p107 or p130 to cyclin/CDK2 complexes is mutually exclusive, and upregulation of p21 leads to abrogation of the p130 or p107 binding to cyclin/CDK2 (Zhu et al., 1995b; Shiyanov et al., 1996). Control of dierentiation through regulation of cell type-speci®c gene expression Pocket proteins are important regulators of certain developmental and dierentiation processes (for a recent extensive review see Yee et al., 1998). In vitro cell dierentiation is typically characterized by the activation of mechanisms of growth arrest that lead to cell cycle exit before genetic programs of dierentiation are turned on. Particularly, regardless of dierentiation inducers, negative cell cycle regulatory pathways converge in the downregulation of E2F-dependent gene expression by formation of pocket protein/E2F repressor complexes in a number of in vitro dierentiating cell types, including dierentiation of skeletal muscle cells (Corbeil et al., 1995; Halevy et al., 1995; Kiess et al., 1995; Shin et al., 1995), neuronal cells (Corbeil et al., 1995), melanoma cells (Jiang et al., 1995), HL60 cells (Ikeda et al., 1996; Smith et al., 1996) and 3T3-L1 adipocytes (Richon et al., 1997) as well as in vivo re-dierentiation of mouse hepatocytes after liver regeneration (Garriga et al., 1998) and retina dierentiation (Kastner et al., 1998; Rampalli et al., 1998). As discussed earlier, most of the E2F-mediated transcriptional repression in this G0 condition seems to be typically exerted by p130/E2F-4 complexes. On the
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other hand, E2F-dependent transcriptional regulation has never been shown to take place in genes involved in cell dierentiation. This suggests that the role of pocket proteins in the control of dierentiation programs involves transcriptional regulation of gene expression by pathways other than, or additional to, the ones used to stop the cell division cycle. Consistently, pRB mutants defective in E2F binding, and therefore, in inducing G1 arrest are still capable of inducing dierentiation-associated ¯at cell morphology in osteosarcoma cells as well as transactivating heterologous promoters from genes induced during dierentiation (Sellers et al., 1998). Experimental evidence implicate pocket proteins as regulatory factors required in cell dierentiation processes. Firstly, pocket protein gene targeting in mice has revealed developmental abnormalities in several tissues. Secondly, ectopic expression of pocket proteins, and particularly of pRB, in certain cell types induces either morphological dierentiation or transcriptional activation of tissue-speci®c genes, for instance, the above mentioned ¯at cell morphology in osteosarcoma cells (Sellers et al., 1998), muscle or adipocyte dierentiation by re-introduction of pocket proteins in pRB7/7 ®broblasts (Gu et al., 1993; Schneider et al., 1994; Chen et al., 1996a; Novitch et al., 1996), neuronal dierentiation of PC12 cells (Kranenburg et al., 1995), and keratinocyte differentiation markers in HaCaT cells (Paramio et al., 1998). Moreover, pRB anti-sense oligonucleotides block in vitro muscle dierentiation of C2 myoblasts (Kobayashi et al., 1998). Thirdly, pocket proteins, pRB being the family member most extensively analysed, have been found to associate directly with a number of proteins with tissue-speci®c activities (Table 1). Some of these proteins are transcription factors involved in the regulation of gene expression during dierentiation processes: (i) pRB, and p107 in pRB7/7 cells, associate with MyoD and myogenin during skeletal muscle dierentiation and activate the expression of muscle speci®c genes (Gu et al., 1993; Schneider et al., 1994; Novitch et al., 1996); (ii) muscle dierentiation also involves association of the transcription factors HBP1 with pRB and p130 (Lavender et al., 1997; Tevosian et al., 1997) and p202 with pRB (Choubey and Lengyel, 1995; Choubey and Gutterman, 1997; Datta et al., 1998); (iii) pRB binds to the related transcription factors C/EBP and NF-IL6 in adipocytes and promotes adipocyte dierentiation (Chen et al., 1996a,b); (iv) the nuclear matrix protein NRP/B, which is required for neuronal dierentiation, forms complexes with pRB (Wiggan et al., 1998); and (v) pRB binding to AP2 complexes activate the AP2dependent transcription of E-cadherin, an intercellular adhesion protein involved in cell polarity and dierentiation of epithelial cells (Batsche et al., 1998). Furthermore, and with uncertain tissue speci®city but with a possible homeotic role, the three pocket proteins associate with paired-like homeodomain transcription factors, including MHox, Chx10, Alx3, and Pax3 (Wiggan et al., 1998). In these studies, Pax3-dependent transcription was shown to be repressed by pocket proteins. Finally, association of pRB with the related transcriptional co-activators BRG1 and hBRM (Dunaief et al., 1994; Singh et al., 1995; Strober et al., 1996), with the transcriptional co-repressor Id2
(Iavarone et al., 1994), and with the thyroid hormone receptor-coactivator Trip230 (Chang et al., 1997) have a less clear role in dierentiation but may be signi®cant in certain homeotic and tissue-speci®c growth processes. In some cases, dierential ability of pocket proteins to bind to some of these transcription factors correlates with a particular functional involvement in the cell dierentiation process. Thus, although p107 and/or p130 can partially substitute for pRB absence in regulating gene expression by myogenic transcription factors, they do not normally do so in wild type pRB cells and pRB is unique in regulating late markers of muscle dierentiation, such as myosin heavy chain expression (Novitch et al., 1996). Consistent with this, pRB de®ciencies in mice lead to defects in skeletal muscle development, among other tissues, while p107 and/or p130 de®ciencies show no abnormalities in this tissue. The unique ability of pRB to complex with myogenic transcription factors such as MyoD and myogenin in vivo, may be an explanation for this dierent functional behavior. In other cases, dierential binding abilities among pocket proteins have just not been assessed. The interaction of pocket proteins with transcription factors involved in dierentiation takes place upon cell cycle exit, thus indicating that hypophosphorylated forms of pocket proteins are involved. Since no cyclin/CDK activities target pocket proteins in dierentiating cells, these associations may not be regulated by phosphorylation during the differentiation process. However, pocket protein hypophosphorylation may be a prerequisite, as exempli®ed by the fact that MyoD is present in proliferating myoblasts but only binds to pRB when cells have exited the cell cycle (Gu et al., 1993). It remains to be analysed if the cell cycle exit-speci®c phosphorylation on p130 has a role in the association of p130 with some of these transcription factors. Regulation of pocket protein levels do seem to play a role in the coordination of pocket protein interactions during some dierentiation processes. Thus, cell cycle exit-associated accumulation of p130 results in abundant p130/E2F-4 complexes that repress transcription of cell cycle genes as a ®rst step in dierentiation, although pRB/E2F complexes have also been detected in some cases (Corbeil et al., 1995; Halevy et al., 1995; Ikeda et al., 1996; Rampalli et al., 1998). The abundance of these pocket protein/E2F complexes, not only allows cell cycle exit, but also, may confer reversibility to the dierentiation process, at least in the early stages. While the involvement of pocket proteins in inducing dierentiation steps rely on punctual biochemical data in many cases, skeletal muscle dierentiation has been the most extensively studied process in this respect, being currently clear that myogenesis involves a cascade of transcriptional events tightly regulated by interdependent myogenic transcription factors (reviewed in Molkentin and Olson, 1996; Rawls and Olson, 1997) and, at least in part, by the coordinated action of pRB and p130 (reviewed in Yee et al., 1998). In this respect, pRB upregulation might trigger a switch from an early reversible phase mediated by dierentiation-repressing factors, such as HBP1, to terminal dierentiation (Shih et al., 1998).
The pRB family in negative cell growth control X GranÄa et al
The biochemistry and biology of pocket proteins tested by their knockouts Pocket proteins do share a number of upstream regulatory mechanisms and also seem to regulate a common subset of cellular targets. For instance, the three proteins are hyperphosphorylated in mid-to-late G1 most likely by cyclin/CDK complexes. However, these common upstream pathways have profound dierent eects on the fate of the three pocket proteins. Although pocket proteins share the ability of interacting with various common cellular proteins, a number of interactions seem to be speci®c for each particular pocket protein. p107 and p130 are structurally more closely related and, consistent with this, there are a number of proteins or protein complexes that interact with them, which do not interact with pRB. These dierences are likely to re¯ect the dierent cellular function of each pocket protein, while the similarities suggest that all pocket proteins might act coordinately to regulate a common set of cellular targets. In addition, we should also consider that some functions of pocket proteins might be redundant, and that dierent pocket proteins might be capable of carrying out the same cellular function in dierent cell types Phenotypes of pRB family mutant mouse strains Gene targeting experiments in mice have begun to delineate clear dierences among pocket proteins. However, these experiments have also indicated that in the absence of a particular pocket protein mechanisms of compensation by the remaining pocket proteins exist (for a more detailed review of these studies see Mulligan and Jacks, 1998). The most severe phenotype observed from analysis of the mouse mutant strains lacking the function of a pRB family member is caused by disruption of the RB gene. Mice lacking functional pRB die during embryogenesis with defects in haematopoiesis, neurogenesis and defects in liver and lens development (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992; Morgenbesser et al., 1994). Although milder, similar abnormalities were found in pRB chimeric mice partially composed of pRB-de®cient cells, which are rescued from the pRB-de®cient lethality and display extensive contribution of pRB7/7 cells in adult tissues (Maandag et al., 1994; Williams et al., 1994). These studies indicate that the function of pRB in certain cell types is essential for their normal development and that neither p107 nor p130 can compensate for pRB absence. In contrast, disruption of either p107 or p130 genes leads to no detectable developmental defect (Cobrinik et al., 1996; Lee et al., 1996). Interestingly, double p1077/7 p1307/7 mice die shortly after birth because of breathing diculties, which arise from a defect in endochondral bone formation. The defect in endochondral bone formation aects long bones and ribs, and is due to excessive chondrocyte proliferation (Cobrinik et al., 1996). These results indicate that either p107 or p130 or both are essential for cell cycle exit in this cell type and that this function cannot be compensated by pRB or by any other as to yet unknown member of this family. Moreover, in contrast to pRB+/7 animals, which show normal retinal
development, chimeras partially composed of pRB7/7 cells in a p1077/7 animal context undergo retinal dysplasia already detectable during fetal development (Robanus-Maandag et al., 1998) and pRB+/7 p1077/7 animals do so during adulthood (Lee et al., 1996), suggesting that sporadic loss of the remaining pRB allele generates these lesions in the absence of p107. Since no other combination of disruption of pRB family members exhibit this phenotype, it appears that either pRB, p107 or both are essential for normal development of mouse retina and p130 cannot compensate for their loss (Lee et al., 1996). The involvement of pRB in lens development was studied in pRB de®cient embryos and in transgenic mice expressing papillomavirus E7 protein (which blocks the function of the pRB family) in the developing lens. Inactivation of pRB function resulted in unchecked proliferation, inhibition of dierentiation and apoptosis. Apoptosis was dependent on a functional p53, since pRB7/7 p537/7 embryos, transgenic mice expressing papillomavirus E7 and E6 (which inactivates p53 function) proteins or transgenic mice expressing the E7 protein in a p537/7 background did not exhibite apoptosis in lens. In regard to tumor susceptibility in pocket proteinde®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 only develop thyroid and pituitary tumors. Interestingly, retinoblastomas, which primarily harbor pRB mutations in humans, are never observed in the pRB+/7 animals (Jacks et al., 1992; Hu et al., 1994; Morgenbesser et al., 1994), which has been attributed to dierences between human and mouse retina. Pituitary tumors are composed of pRB7/7 cells indicating loss of the wild type allele. Conversely, introduction of a wild type pRB transgene in these pRB+/7 mice completely prevents tumor formation (Riley et al., 1997). Tumor suppression by pRB can also be assessed using the pRB-de®cient chimeras which, in agreement with previous results, only undergo pituitary tumorigenesis, being the tumors analyzed almost exclusively formed by pRB7/7 cells (Maandag et al., 1994; Williams et al., 1994). This indicates that the second mutational event found in the wild type-pRB allele of pituitary tumors from pRB+/7 mice (Jacks et al., 1992; Hu et al., 1994) is critical for tumor formation. Consistent with this, the age of chimeric animals in which pituitary tumors were detected was signi®cantly reduced compared to pRB+/7 animals. On the other hand, the fact that pRB gene targeting in mice only provokes thyroid and pituitary tumors does not rule out that pRB loss can also contribute to carcinogenesis in other tissues. Thus, in a pRB-de®cient setting, the time before mice die from pituitary cancer may be too short to allow for additional mutations in other genes, which are known to be required in the multi-step character of carcinogenesis in many tissues. In support of this, transgenic mice expressing E7 in lens displayed tumors only in the absence of functional p53 (Howes et al., 1994; Morgenbesser et al., 1994; Pan and Griep, 1994). Moreover, induction of tumors in the brain choroid plexus epithelium demonstrated a cooperation in tumorigenesis by inactivation of p53 and the pRB family (Symonds et al., 1994). Therefore, the murine
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pituitary gland, similar to the retina in humans, might be a tissue highly susceptible to cell transformation when pRB function is impaired, whereas other tissues may require further mutational alterations, such as loss of p53 function. Similarly, the above mentioned retinal dysplasia in pRB-chimeric, p1077/7 mouse fetuses is followed by the formation of retinoblastomas in adulthood (Robanus-Maandag et al., 1998), suggesting again that additional alterations are limiting for the tumor promotion of these pRB/p107-de®cient retinal pre-neoplastic lesions. Since neither pRB+/7 mice nor pRB-de®cient chimeric animals display retinal tumors (Jacks et al., 1992; Hu et al., 1994; Maandag et al., 1994; Williams et al., 1994), p107 might have a role as a tumor suppressor in the retina. However, the functional implications of p107 in carcinogenesis are still unclear because pituitary tumors are abrogated in pRB+/7 p1077/7 compared to pRB+/7 mice (Lee et al., 1996). Indeed, in contrast to pRB, neither heterozygous nor homozygous deletions of p107, p130 or both in mice lead to tumor formation in any tissue (Cobrinik et al., 1996; Lee et al., 1996), which is in agreement with the fact that these genes have rarely been found mutated in human cancer. Nevertheless, in support of a role for p130 and p107 as tumor suppressors, it has been shown that a growth advantage conferred by SV40 large T-antigen in MEFs requires inactivation of both p130 and p107, a feature that might contribute to T-antigen-mediated cell transformation (Christensen and Imperiale, 1995; Zalvide and DeCaprio, 1995; Stubdal et al., 1997). E2F-1 is a downstream target of pRB in E2F-1 mediated induction of S phase, apoptosis and tumorigenesis As we have discussed in previous sections, pRB negative function on cell proliferation is thought to be mediated, at least in part, by repression of members of the E2F family of transcription factors. Among E2F family members, E2F-1 is speci®cally targeted by pRB, but not by p107 or p130. The role of E2F-1 as a downstream target of pRB during tumorigenesis is starting to be understood. In pRB+/7 E2F-17/7 mice the frequency of thyroid and pituitary tumors is reduced compared to the frequency in pRB+/7 mice harboring wild type E2F-1, which demonstrates that E2F-1 is involved in the development of tumors that arise in pRB+/7 mice (Yamasaki et al., 1998). The role of E2F-1 as a downstream target of pRB in cell cycle control and apoptosis in vivo has been studied during embryonic development and in epithelial brain tumors (Pan et al., 1998; Tsai et al., 1998). Double pRB7/7 E2F7/7 embryos die by E17, later than pRB7/7 embryos which die between E14 and E15, with anemia and defective muscle and lung development. Since E2F17/7 mice are viable (Field et al., 1996; Yamasaki et al., 1996), these results indicate that although E2F-1 function is required for some of the defects observed in pRB7/7 embryos, it is not the sole essential target of pRB during mouse embryonic development. Other essential targets of pRB might include other E2F family members and/or transcription factors involved in cell dierentiation. In this context, transgenic pRB7/7 fetuses expressing an N-terminal truncated pRB mutant, which retains the ability to bind to E2F, have a delayed prenatal death but still show abnormalities in
erythropoiesis, skeletal muscle and nervous system while the wild type pRB transgene allows normal development of these tissues (Riley et al., 1997). Moreover, this pRB mutant transgene does not prevent, as wild type pRB does, the appearance of thyroid tumors in pRB+/7 mice (Riley et al., 1997). These studies suggest that at least some of the eects of pRB absence in mice are not the result of an impairment of the pRB targeting function on E2F-1. Double mutant pRB7/7 E2F-17/7 embryos exhibit reduced apoptosis and S phase entry in certain tissues, such as lens and CNS, when compared with their counterpart pRB7/7 tissues, indicating that pRB normally represses E2F-1-mediated S phase induction and apoptosis in these tissues. The involvement of p53 in these apoptotic scenarios was indicated by the ®nding that p53 DNA-binding activity and the expression of a p53 target gene, the p21CIP1 gene, are reduced in the pRB7/7 E2F-17/7 double mutant and wild type brains, while they are increased in pRB7/7 brains (Pan et al., 1998; Tsai et al., 1998). Similar conclusions were reached by using a transgenic model in which expression of a N-terminal fragment of T antigen is targeted to the choroid plexus. Expression of this T antigen (T121) mutant blocks the pRB family, but does not block p53. Aggressive epithelial brain tumors are induced by this T antigen mutant in a p537/7 background. In a p53+/+ background, p53 target genes are upregulated instead and apoptosis occurs, resulting in tumor growth attenuation (Symonds et al., 1994). 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. However, no tumors generate because E2F-1 absence also impairs S phase induction (Pan et al., 1998; Tsai et al., 1998). Altogether, these ®ndings suggest a model in which pRB restricts induction of both S phase and p53dependent apoptosis by E2F-1. Cellular and molecular consequences of disruption of the function of pRB family members The eects of disrupting the function of each pRB family member as well as their combinations have been studied in detail in mouse embryonic ®broblasts (MEFs) and, in the case of p130 and/or p107, also in peripheral T lymphocytes. Firstly, these experiments have shown both dierences in the time period required to reach S phase upon serum re-stimulation of MEFs and dierences in the patterns of expression of a number of E2F-regulated genes. Secondly, these experiments have also shown functional compensation among pocket proteins (Almasan et al., 1995; Lukas et al., 1995a; Herrera et al., 1996; Hurford et al., 1997; Mulligan et al., 1998). In respect to the dierences in the expression of E2F-regulated genes, MEFs lacking functional pRB, but not p107 or p130, exhibit derepression of a subset of E2F-target genes in G0/G1, including the cyclin E and p107 genes. Compensation among pocket proteins is suggested by the fact that only MEFs lacking both p107 and p130 exhibit deregulation of a dierent subset of E2F-target genes in G0/G1, including B-myb, cdc2, E2F-1, TS, ribonucleotide reductase subunit M2 (RRM2), cyclin A and DHFR genes (Hurford et al., 1997). The fact that p130 is expressed at high levels in
The pRB family in negative cell growth control X GranÄa et al
quiescent wild type cells and is the major pocket protein associated with E2F in these cells, suggests that lack of p130 is compensated by p107, but in the absence of both proteins pRB cannot repress at least some of the E2F-target genes normally repressed by p130. This is further supported by the ®nding that pRB and p130/p107 seem to regulate dierent E2F-target genes. Analysis of the expression of E2F-target genes in p1077/7 p1307/7 T lymphocytes exhibited similar elevated expression of B-myb, cdc2, cyclin A2 and RRM2 mRNAs when compared with p107+/7 p1307/7 and p1077/7 p130+/7 T lymphocytes (Mulligan et al., 1998). In murine T lymphocytes lacking p130, p107 levels are elevated, and the E2F DNA binding activity detected contains p107 instead of the absent p130. In p1077/7 p1307/7 peripheral T lymphocytes higher levels of free E2F are detected than in p1307/7 cells, but most of the remaining E2F DNA binding activity contains pRB. Interestingly, a new E2F complex containing a protein dierent from the known members of the pRB family seems to be induced in the absence of p107 and p130. A similar complex was also detected in p1077/7 p1307/7 MEFs (Hurford et al., 1997; Mulligan et al., 1998). Thus, clear compensation mechanisms exist, such that particular functions of each pocket protein are preserved in their absence. p130 and p107 seem to compensate for each other quite eciently, and this might explain the lack of abnormalities in the p1077/7 and p1307/7 animals. This conclusion is in agreement with the higher structural similarity between these two proteins when compared to pRB, and the fact that p107 and p130 interact with a common subset of proteins, which do not interact with pRB or exhibit lower anity for pRB. The much severe phenotypes seen in pRB7/7 mice, indicate that a number of essential functions of pRB, cannot be performed by either p107 or p130. This might be due to the inability of p107 or p130 to interact with a number of pRB targets such as E2F family members 1, 2 and 3, as well as other transcription factors. Alternatively, the levels of expression of p130 or p107 in the aected tissues might not be sucient to substitute for the absence of pRB. The higher compensatory eciency between p130 and p107, together with pRB ability to compensate at least partially for p107/p130 loss might also explain the absence of tumors in p107+/7, p130+/7, p107+/7 p1307/7 and p1077/7 p130+/7 animals. Signi®cance of the regulation of pocket proteins in the negative control of cell growth The wide range of pocket protein interactions with several cellular proteins (Table 1), together with the ubiquitous nature of some mechanisms of pocket protein action, such as modulation of E2F transcription factors, have indicated that the role of pocket proteins is not restricted to cell types or to cell functions. Moreover, diverse aspects of pocket protein function are tightly regulated primarily by two simple mechanisms: phosphorylation status and protein levels. The conserved mechanisms of pocket protein regulation in diverse cell types and growth instances is in contrast to the dramatically dierent consequences that seem to depend on the epigenetic context of the cell.
For instance, the cell cycle exit pattern of pocket proteins triggers a dierentiation program only when transcription factors and other mediators required for that program are present. Such is the case when expression of MyoD or C/EBP transcription factors only promote dierentiation in pRB7/7 ®broblasts if pRB is re-introduced in the cells. This reasoning could be extended to many other scenarios. Therefore, pocket proteins could be de®ned as competence factors which, as discussed in this review, will allow a number of diverse cell growth and dierentiation transitions. It is assumed that a universal mechanism of cell cycle exit involves transcriptional silencing of E2Fresponsive genes by pocket proteins. Thus, a multitude of negative growth signals converge by dierent mechanisms in the inactivation of cyclin/CDK activities leading to the hypophosphorylation of pocket proteins (reviewed by GranÄa and Reddy, 1995; Mayol and GranÄa, 1998; Yee et al., 1998). In this scenario, complex formation with E2F is mostly regulated by the phosphorylation state of pocket proteins. Silencing of genes involved in cell cycle progression will thus be determined by the active repression of pocket protein/ E2F complexes or by the absence of free, transcriptionally-activating E2F proteins due to pocket protein sequestration. Since cell growth regulatory pathways in G1 are complex and often involve feedback regulatory loops, determining pocket protein cooperativity during cell cycle exit is dicult. In this context, the presence of pRB/E2F and p107/E2F complexes in proliferating cells, but not p130/E2F-4, suggests that the involvement of E2F in the passage through the R point is regulated by pRB and/or p107 rather than by p130. It is still unclear if the cell cycle exit-speci®c phosphorylation of p130, leading to its accumulation in the form of p130/E2F-4 complexes, is a triggering event of cell cycle exit or a consequence of it. Once in quiescence, p130/E2F complexes persist and, in fact, are typically found in many dierentiated adult tissues. During cell dierentiation, keeping E2Fdependent cell cycle genes silent seems to be a prerequisite for the expression of dierentiation genes (Rao et al., 1994; Skapek et al., 1995; Wang et al., 1996; Guo and Walsh, 1997). Considering that the repressor activity of p130/E2F-4 complexes is possibly a requirement for G1 re-entrance from G0, their presence in quiescent cells may also have the signi®cance of conferring reversibility to the cell cycle exit program. In the context of cell dierentiation, p130/E2F complexes have been assigned to play a role in an early, dierentiation-independent phase of cell 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 normally carried out by p130, because pRB absence, conversely, leads to a defect in terminal dierentiation that causes cell cycle exit reversibility (Schneider et al., 1994; Novitch et al., 1996; Zacksenhaus et al., 1996). The existence of certain tissue-speci®c transcription factors involved in negative regulation of gene expression during dierentiation, such as HBP1 and p202 in muscle and CHOP in adipocytes, is in support of this reversible early phase (reviewed in Yee et al., 1998). In particular, HBP1 requires binding to pocket proteins for its negative function in muscle differentiation and, in fact, increasing levels of pRB reverse this eect and allow myogenic gene expression (Shih et al.,
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1998). These data have led to propose that upregulation of pRB protein levels, which is observed during in vitro dierentiation of skeletal muscle cells, would trigger the transition from an early reversible phase to terminal dierentiation (reviewed in Yee et al., 1998). The transcription factors HBP-1, p202 and CHOP not only repress dierentiation gene expression but also induce cell cycle exit when overexpressed (Barone et al., 1994; Tevosian et al., 1997; Datta et al., 1998), and in the case of p202, direct inhibition of either E2F-1 or E2F-4 transcriptional activity has been reported (Choubey and Lengyel, 1995; Choubey and Gutterman, 1997). These data indicate that these transcription factors have a dual role in the early reversible phase of dierentiation: induction of cell cycle exit, perhaps in cooperation with pocket protein/E2F repressor complexes, and delay of dierentiation gene expression until terminal dierentiation inducers (such as pRB) are activated. However, the biological signi®cance of a pocket protein-dependent reversible period during dierentiation where cells exercise an option to undergo dierentiation is still unclear. Upon cell cycle re-entry, it is striking that only p130/ E2F complexes are found in the early, pre-R point passage phase. It is conceivable that E2F transcriptional repression during this stage underlies a complex network of growth factor transduction pathways that need to be coordinately activated for a proper cell cycle re-entrance (reviewed in Mayol and GranÄa, 1998). As an example, one of the genes thought to be repressed 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., 1993; Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994; DeGregori et al., 1995; Kowalik et al., 1995). Interestingly, induction of apoptosis by E2F-1 is separable from its transcriptional transactivation capacity but dependent on DNA binding (Phillips et al., 1997). A transactivation domain-defective E2F mutant relieves, probably by competition for DNA binding sites, the transcriptional repressor activity of p130/E2F-4 complexes in quiescent cells. Altogether these results suggest that suppression of E2F-mediated transcriptional repression accounts for, at least in part, S phase induced apoptosis by E2F-1. In this context, p130/E2F-4-mediated repression of genes like the E2F1 gene may perform the function of keeping key regulators of R point passage silent until a number of G1 regulatory pathways have been activated and integrated into a single R point passage decision. In the absence of p130-mediated transcriptional repression, that is, in post-mitotic G1 proliferating cells, pRB assumes the role of controlling E2F function in G1 by regulation of E2F-3, but not other E2F family members, at least in Ref 52 cells (Leone et al., 1998). The consequences, in terms of gene expression, of whether E2F is regulated by p130 or by pRB during early G1 are not known. Moreover, p130/E2F repressor complexes have the responsibility of maintaining a pool of E2F proteins ready to trigger E2Fdependent transcription upon cyclin/CDK activation.
The fact that p130/E2F-4 complexes repress the expression of the pRB-associated E2Fs (E2F-1 and E2F-2) also plays a role in conferring order to the coordinated action of pocket proteins during G1. In this case, pRB action on E2F-dependent gene expression would be dependent on the previous action of p130. Finally, by extension, p107 expression in mid G1 will also be dependent on this activating cascade since it is repressed by both p130 and pRB. Therefore, regulation of pocket protein phosphorylation status and levels during G1 integrates growth signal transduction pathways by coordinating the interaction of pocket proteins with E2F and other transcription factors (see Table 1). This culminates in the passage through the R point and in further progression through S phase. Precise details on how pocket proteins regulate cell growth and dierentiation processes are thus starting to be understood. Although the coordination in the interactions with cellular proteins during cell growth situations is yet to be precisely grasped in many aspects, it seems clear that the presence of a particular phosphorylated pocket protein form in a given cell type, growth stage, and cellular compartment has determinant consequences in the physiology of the cell. The above discussed cellular processes seem to be dependent on the ability of pocket proteins to coordinate a multitude of regulatory processes acting as competence factors to allow or impede certain cell growth and/or dierentiation transitions.
Note added in proof After this review article was submitted to the publishers two papers were published describing the generation of p1307/7 and p1077/7 mutant mice in enriched Balb/cJ backgrounds (LeCourter et al., 1998a: Development 125, 4669 ± 4679; LeCourter et al., 1998b: Mol. Cell. Biol. 18, 7455 ± 7465). Balb/cJ mice lacking p130 died between embryonic days 11 and 13 due to developmental abnormalities, which in contrast with the apparent lack of abnormalities of the previously obtained mutant animals generated in a dierent genetic background. Balb/cJ mice lacking p107 are viable but exhibit impaired growth. Interestingly both mutant animals revert to a wild type phenotype when crossed with C57BL/6J mice, suggesting the existence of modi®er genes that have epistatic relationships with p130 and p107. A second study describing the association of HDAC1 with p130 has also been published (Stiegler et al., 1998: Cancer Res. 58, 5049 ± 5052). Acknowledgements We thank Sushil Rane, Ana LimoÂn and Matilde ParrenÄo for critical reading of the manuscript and suggestions. We apologize to colleagues whose work has not been cited, or cited indirectly through other articles, due to space limitations. XM and JG were supported by fellowships from DireccioÂn General de Investigacio n Cientõ ®ca y TeÂcnica (Ministerio de EducacioÂn y Cultura, Spain). Work in XG laboratory was supported by a grant from the National Institute of General Medical Sciences, NIH (GM54894).
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References Adams JA, McGlone ML, Gibson R and Taylor SS. (1995). Biochem., 34, 2447 ± 2454. Adams PD, Sellers WR, Sharma SK, Wu AD, Nalin CM and Kaelin WG. (1996). Mol. Cell. Biol., 16, 6623 ± 6633. Alevizopoulos K, Vlach J, Hennecke S and Amati B. (1997). EMBO. J., 16, 5322 ± 5333. Almasan A, Yin Y, Kelly RE, Lee EH, Bradley A, Li W, Bertino JR and Wahl GM. (1995). Proc. Natl. Acad. Sci. USA, 92, 5436 ± 5440. Barone MV, Crozat A, Tabaee A, Philipson L and Ron D. (1994). Genes Dev., 8, 453 ± 464. Bartek J, Bartkova J and Lukas J. (1996). Curr. Opin. Cell. Biol., 8, 805 ± 814. Batsche E, Muchardt C, Behrens J, Hurst HC and Cremisi C. (1998). Mol. Cell. Biol., 18, 3647 ± 3658. Beijersbergen RL, Carlee L, Kerkhoven RM and Bernards R. (1995). Genes Dev., 9, 1340 ± 1353. Belbrahem A, Goddenkent D and Mittnacht S. (1996). Exp. Cell. Res., 225, 286 ± 293. Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B, Eilers M, Hatzigeorgiou A and Jansen-Durr P. (1996). Mol. Cell. Biol., 16, 3401 ± 3409. Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ and Kouzarides T. (1998). Nature, 391, 597 ± 601. Buchkovich K, Duy LA and Harlow E. (1989). Cell, 58, 1097 ± 1105. Cartwright P, Muller H, Wagener C, Holm K and Helin K. (1998). Oncogene, 17, 611 ± 623. Castano E, Kleyner Y and Dynlacht BD. (1998). Mol. Cell. Biol., 18, 5380 ± 5391. Cavanaugh AH, Hempel WM, Taylor LJ, Rogalsky V, Todorov G and Rothblum LI. (1995). Nature, 374, 177 ± 180. Chang KH, Chen Y, Chen TT, Chou WH, Chen PL, Ma YY, Yang-Feng TL, Leng X, Tsai MJ, O'Malley BW and Lee WH. (1997). Proc. Natl. Acad. Sci. USA, 94, 9040 ± 9045. Chen PL, Riley DJ, Chen YM and Lee WH. (1996a). Genes Dev., 10, 2794 ± 2804. Chen PL, Riley DJ, Chen-Kiang S and Lee WH. (1996b). Proc. Natl. Acad. Sci. USA, 93, 465 ± 469. Chen PL, Scully P, Shew JY, Wang JY and Lee WH. (1989). Cell, 58, 1193 ± 198. Chittenden T, Livingston DM and DeCaprio JA. (1993). Mol. Cell. Biol., 13, 3975 ± 3983. Choubey D and Gutterman JU. (1997). Oncogene, 15, 291 ± 301. Choubey D and Lengyel P. (1995). J. Biol. Chem., 270, 6134 ± 6140. Christensen JB and Imperiale MJ. (1995). J. Virol., 69, 3945 ± 3948. Clarke AR, Maandag ER, van Roon M, van der Lugt NM, van der Valk M, Hooper ML, Berns A and te Riele H. (1992). Nature, 359, 328 ± 330. Clurman BE, Shea RJ, Thress K, Groudine M and Roberts JM. (1996). Genes Dev., 10, 1979 ± 1990. Cobrinik D, Lee MH, Hannon G, Mulligan G, Bronson RT, Dyson N, Harlow E, Beach D, Weinberg RA and Jacks T. (1996). Genes Dev., 10, 1633 ± 1644. Cobrinik D, Whyte P, Peeper DS, Jacks T and Weinberg RA. (1993). Genes Dev., 7, 2392 ± 2404. Connell-Crowley L, Harper JW and Goodrich DW. (1997). Mol. Biol. Cell., 8, 287 ± 301. Corbeil HB, Whyte P and Branton PE. (1995). Oncogene, 11, 909 ± 920. Dalton S. (1992). EMBO J., 11, 1797 ± 1804. Datta B, Min W, Burma S and Lengyel P. (1998). Mol. Cell. Biol., 18, 1074 ± 1083. DeCaprio JA, Ludlow JW, Lynch D, Furukawa Y, Grin J, Piwnica-Worms H, Huang CM and Livingston DM. (1989). Cell, 58, 1085 ± 1095.
DeGregori J, Kowalik T and Nevins JR. (1995). Mol. Cell. Biol., 15, 4215 ± 4224. Deluca A, Maclachlan TK, Bagella L, Dean C, Howard CM, Claudio PP, Baldi A, Khalili K and Giordano A. (1997). J. Biol. Chem., 272, 20971 ± 20974. Diehl JA, Zindy F and Sherr CJ. (1997). Genes Dev., 11, 957 ± 972. Dou QP, Markell PJ and Pardee AB. (1992). Proc. Natl. Acad. Sci. USA, 89, 3256 ± 3260. Dowdy SF, Hinds PW, Louie K, Reed SI, Arnold A and Weinberg RA. (1993). Cell, 73, 499 ± 511. Dunaief JL, Strober BE, Guha S, Khavari PA, Alin K, Luban J, Begemann M, Crabtree GR and Go SP. (1994). Cell, 79, 119 ± 130. Dynlacht BD. (1995). Nature, 374, 114 ± 114. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato J and Livingston DM. (1993). Cell, 73, 487 ± 497. Ewen ME, Xing YG, Lawrence JB and Livingston DM. (1991). Cell, 66, 1155 ± 1164. Ferreira R, Magnaghi-Jaulin L, Robin P, Harel-Bellan A and Trouche D. (1998). Proc. Natl. Acad. Sci. USA, 95, 10493 ± 10498. Field SJ, Tsai FY, Kuo F, Zubiaga AM, Kaelin WG, Jr., Livingston DM, Orkin SH and Greenberg ME. (1996). Cell, 85, 549 ± 561. Garriga J, Limon A, Mayol X, Rane SG, Albrecht JH, Reddy EP, AndreÂs V and GranÄa X. (1998). Biochem. J., 333, 645 ± 654. Gaubatz S, Wood JG and Livingston DM. (1998). Proc. Natl. Acad. Sci. USA, 95, 9190 ± 9195. Geng Y, Eaton EN, Picon M, Roberts JM, Lundberg AS, Giord A, Sardet C and Weinberg RA. (1996). Oncogene, 12, 1173 ± 1180. GranÄa X and Reddy EP. (1995). Oncogene, 11, 211 ± 219. Gu W, Schneider JW, Condorelli G, Kaushal S, Mahdavi V and Nadal GB. (1993). Cell, 72, 309 ± 324. Guo K and Walsh K. (1997). J. Biol. Chem., 272, 791 ± 797. Halevy O, Novitch BG, Spicer DB, Skapek SX, Rhee J, Hannon GJ, Beach D and Lassar AB. (1995). Science, 267, 1018 ± 1021. Hannon GJ, Demetrick D and Beach D. (1993). Genes Dev., 7, 2378 ± 2391. Hauser PJ, Agrawal D, Chu BK and Pledger WJ. (1997). J. Biol. Chem., 272, 22954 ± 22959. Herrera RE, Sah VP, Williams BO, Makela TP, Weinberg RA and Jacks T. (1996). Mol. Cell. Biol., 16, 2402 ± 2407. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI and Weinberg RA. (1992). Cell, 70, 993 ± 1006. Hiyama H, Iavarone A and Reeves SA. (1998). Oncogene, 16, 1513 ± 1523. Horton LE, Qian Y and Templeton DJ. (1995). Cell Growth Dier., 6, 395 ± 407. Hoshikawa Y, Mori A, Amimoto K, Iwabe K and Hatakeyama M. (1998). Proc. Natl. Acad. Sci. USA, 95, 8574 ± 8579. Howes KA, Ransom N, Papermaster DS, Lasudry J, Albert DM and Windle JJ. (1994). Genes Dev., 8, 1300 ± 1310. Hsiao KM, McMahon SL and Farnham PJ. (1994). Genes Dev., 8, 1526 ± 1537. Hu N, Gutsmann A, Herbert DC, Bradley A, Lee WH and Lee EY. (1994). Oncogene, 9, 1021 ± 1027. Huet X, Rech J, Plet A, Vie A and Blanchard JM. (1996). Mol. Cell. Biol., 16, 3789 ± 3798. Hurford RK, Cobrinik D, Lee MH and Dyson N. (1997). Genes Dev., 11, 1447 ± 1463. Iavarone A, Garg P, Lasorella A, Hsu J and Israel MA. (1994). Genes Dev., 8, 1270 ± 1284. Ikeda M, Jakoi L and Nevins JR. (1996). Proc. Natl. Acad. Sci. USA, 93, 3215 ± 3220.
The pRB family in negative cell growth control X GranÄa et al
3382
Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA and Weinberg RA. (1992). Nature, 359, 295 ± 300. Jiang HP, Lin J, Young SM, Goldstein NI, Waxman S, Davila V, Chellappan SP and Fisher PB. (1995). Oncogene, 11, 1179 ± 1189. Johnson DG. (1995). Oncogene, 11, 1685 ± 1692. Johnson DG, Ohtani K and Nevins JR. (1994). Genes Dev., 8, 1514 ± 1525. Johnson DG and Schneider-Broussard R. (1998). Front. Biosci., 3, d447 ± 448. Johnson DG, Schwarz JK, Cress WD and Nevins JR. (1993). Nature, 365, 349 ± 352. Johnson LF. (1992). Curr. Opin. Cell. Biol., 4, 149 ± 154. Kastner A, Espanel X and Brun G. (1998). Cell Growth Dier., 9, 857 ± 867. Kato J, Matsushime H, Hiebert SW, Ewen ME and Sherr CJ. (1993). Genes Dev., 7, 331 ± 342. Kiess M, Gill RM and Hamel PA. (1995). Cell. Growth Dier., 6, 1287 ± 1298. Kitagawa M, Higashi H, Jung HK, Suzuki-Takahashi I, Ikeda M, Tamai K, Kato J, Segawa K, Yoshida E, Nishimura S and Taya Y. (1996). EMBO J., 15, 7060 ± 7069. Knudsen ES, Buckmaster C, Chen TT, Feramisco JR and Wang JY. (1998). Genes Dev., 12, 2278 ± 2292. Knudsen ES and Wang JY. (1996). J. Biol. Chem., 271, 8313 ± 8320. Knudsen ES and Wang JY. (1998). Oncogene, 16, 1655 ± 1663. Kobayashi M, Yamauchi Y and Tanaka A. (1998). Exp. Cell. Res., 239, 40 ± 49. Kowalik TF, DeGregori J, Schwarz JK and Nevins JR. (1995). J. Virol., 69, 2491 ± 2500. Kranenburg O, De GR, Van der Eb, AJ and Zantema A. (1995). Oncogene, 10, 87 ± 95. Lacy S and Whyte P. (1997). Oncogene, 14, 2395 ± 2406. Larminie CG, Cairns CA, Mital R, Martin K, Kouzarides T, Jackson SP and White RJ. (1997). EMBO J., 16, 2061 ± 2071. Lavender P, Vandel L, Bannister AJ and Kouzarides T. (1997). Oncogene, 14, 2721 ± 2728. Lee EY, Chang CY, Hu N, Wang YC, Lai CC, Herrup K, Lee WH and Bradley A. (1992). Nature, 359, 288 ± 294. Lee MH, Williams BO, Mulligan G, Mukai S, Bronson RT, Dyson N, Harlow E and Jacks T. (1996). Genes Dev., 10, 1621 ± 1632. Lees E, Faha B, Dulic V, Reed SI and Harlow E. (1992). Genes Dev., 6, 1874 ± 1885. Lees JA, Buchkovich KJ, Marshak DR, Anderson CW and Harlow E. (1991). EMBO J., 10, 4279 ± 4290. Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams RS and Nevins JR. (1998). Genes Dev., 12, 2120 ± 2130. Li Y, Graham C, Lacy S, Duncan AM and Whyte P. (1993). Genes Dev., 7, 2366 ± 2377. Lin BT, Gruenwald S, Morla AO, Lee WH and Wang JY. (1991). EMBO J., 10, 857 ± 864. Ludlow JW and Nelson DA. (1995). Semin. Cancer Biol., 6, 195 ± 202. Lukas J, Bartkova J, Rohde M, Strauss M and Bartek J. (1995a). Mol. Cell. Biol., 15, 2600 ± 2611. Lukas J, Herzinger T, Hansen K, Moroni MC, Resnitzky D, Helin K, Reed SI and Bartek J. (1997). Genes Dev., 11, 1479 ± 1492. Lukas J, Muller H, Bartkova J, Spitkovsky D, Kjerul AA, Jansen DP, Strauss M and Bartek J. (1994). J. Cell. Biol., 125, 625 ± 638. Lukas J, Parry D, Aagaard L, Mann DJ, Bartkova J, Strauss M, Peters G and Bartek J. (1995b). Nature, 375, 503 ± 506. Lundberg AS and Weinberg RA. (1998). Mol. Cell. Biol., 18, 753 ± 761. Luo RX, Postigo AA and Dean DC. (1998). Cell, 92, 463 ± 473.
Maandag EC, van der Valk M, Vlaar M, Feltkamp C, O'Brien J, van Roon M, van der Lugt N, Berns A and te Riele H. (1994). EMBO J., 13, 4260 ± 4268. Magnaghi-Jaulin L, Groisman R, Naguibneva I, Robin P, Lorain S, Le Villain JP, Troalen F, Trouche D and HarelBellan A. (1998). Nature, 391, 601 ± 605. Mayol X, Garriga J and GranÄa X. (1995). Oncogene, 11, 801 ± 808. Mayol X, Garriga J and GranÄa X. (1996). Oncogene, 13, 237 ± 246. Mayol X and GranÄa X. (1997). In: Progress in Cell Cycle Research, Vol. 3. Meijier LGS and Philippe M (ed.). Plenum Press: New York, pp. 157 ± 169. Mayol X and GranÄa X. (1998). Front. Biosci., 3, 11 ± 24. Mayol X, GranÄa X, Baldi A, Sang N, Hu Q and Giordano A. (1993). Oncogene, 8, 2561 ± 2566. Means AL, Slansky JE, McMahon SL, Knuth MW and Farnham PJ. (1992). Mol. Cell. Biol., 12, 1054 ± 1063. Medema RH, Herrera RE, Lam F and Weinberg RA. (1995). Proc. Natl. Acad. Sci. USA, 92, 6289 ± 6293. Mihara K, Cao XR, Yen A, Chandler S, Driscoll B, Murphree AL, T'Ang A and Fung YK. (1989). Science, 246, 1300 ± 1303. Molkentin JD and Olson EN. (1996). Curr. Opin. Genet. Dev., 6, 445 ± 453. Morgenbesser SD, Williams BO, Jacks T and DePinho RA. (1994). Nature, 371, 72 ± 74. Morkel M, Wenkel J, Bannister AJ, Kouzarides T and Hagemeier C. (1997). Nature, 390, 567 ± 568. Mulligan GJ and Jacks T. (1998). Trends Genet., 14, 223 ± 229. Mulligan GJ, Wong J and Jacks T. (1998). Mol. Cell. Biol., 18, 206 ± 220. Neuman E, Flemington EK, Sellers WR and Kaelin WJ. (1994). Mol. Cell. Biol., 14, 6607 ± 6615. Niculescu AB, Chen X, Smeets M, Hengst L, Prives C and Reed SI. (1998). Mol. Cell. Biol., 18, 629 ± 643. Novitch BG, Mulligan GJ, Jacks T and Lassar AB. (1996). J. Cell. Biol., 135, 441 ± 456. Ogris E, Rotheneder H, Mudrak I, Pichler A and Wintersberger E. (1993). J. Virol., 67, 1765 ± 1771. Ohtani K, Degregori J, Leone G, Herendeen DR, Kelly TJ and Nevins JR. (1996). Mol. Cell. Biol., 16, 6977 ± 6984. Ohtani K, DeGregori J and Nevins JR. (1995). Proc. Natl. Acad. Sci. USA, 92, 12146 ± 12150. Ohtani K, Tsujimoto A, Ikeda M and Nakamura M. (1998). Oncogene, 17, 1777 ± 1785. Oswald F, Dobner T and Lipp M. (1996). Mol. Cell. Biol., 16, 1889 ± 1895. Pan H and Griep AE. (1994). Genes Dev., 8, 1285 ± 1299. Pan H, Yin C, Dyson NJ, Harlow E, Yamasaki L and Dyke TV. (1998). Mol. Cell., 2, 283 ± 292. Paramio JM, Lain S, Segrelles C, Lane EB and Jorcano JL. (1998). Oncogene, 17, 949 ± 957. Philips A, Huet X, Plet A, Le Cam L, Vie A and Blanchard JM. (1998). Oncogene, 16, 1373 ± 1381. Phillips AC, Bates S, Ryan KM, Helin K and Vousden KH. (1997). Genes Dev., 11, 1853 ± 1863. Puri PL, Cimino L, Fulco M, Zimmerman C, La Thangue NB, Giordano A, Graessmann A and Levrero M. (1998). Cancer Res., 58, 1325 ± 1331. Qin XQ, Livingston DM, Kaelin Jr WG and Adams PD. (1994). Proc. Natl. Acad. Sci. USA, 91, 10918 ± 10922. Rampalli AM, Gao CY, Chauthaiwale VM and Zelenka PS. (1998). Oncogene, 16, 399 ± 408. Rao SS, Chu C and Kohtz DS. (1994). Mol. Cell. Biol., 14, 5259 ± 5267. Rawls A and Olson EN. (1997). Cell, 89, 5 ± 8. Resnitzky D, Gossen M, Bujard H and Reed SI. (1994). Mol. Cell. Biol., 14, 1669 ± 1679. Resnitzky D and Reed SI. (1995). Mol. Cell. Biol., 15, 3463 ± 3469.
The pRB family in negative cell growth control X GranÄa et al
Richon VM, Lyle RE and McGehee RE. (1997). J. Biol. Chem., 272, 10117 ± 10124. Riley DJ, Liu CY and Lee WH. (1997). Mol. Cell. Biol., 17, 7342 ± 7352. Robanus-Maandag E, Dekker M, van der Valk M, Carrozza ML, Jeanny JC, Dannenberg JH, Berns A and te Riele H. (1998). Genes Dev., 12, 1599 ± 1609. Sardet C, Vidal M, Cobrinik D, Geng Y, Onufryk C, Chen A and Weinberg RA. (1995). Proc. Natl. Acad. Sci. USA, 92, 2403 ± 2407. Schneider JW, Gu W, Zhu L, Mahdavi V and Nadal GB. (1994). Science, 264, 1467 ± 1471. Schulze A, Mannhardt B, Zerfass-Thome K, Zwerschke W and Jansen-Durr P. (1998). J. Virol., 72, 2323 ± 2334. Schulze A, Zerfass K, Spitkovsky D, Middendorp S, Berges J, Helin K, Jansen-Durr P and Henglein B. (1995). Proc. Natl. Acad. Sci. USA, 92, 11264 ± 11268. Schwarz JK, Devoto SH, Smith EJ, Chellappan SP, Jakoi L and Nevins JR. (1993). EMBO J., 12, 1013 ± 1020. Sears R, Ohtani K and Nevins JR. (1997). Mol. Cell. Biol., 17, 5227 ± 5235. Sellers WR, Novitch BG, Miyake S, Heith A, Otterson GA, Kaye FJ, Lassar AB and Kaelin Jr WG. (1998). Genes Dev., 12, 95 ± 106. Shan B and Lee WH. (1994). Mol. Cell. Biol., 14, 8166 ± 8173. Shea RJ, Groudine M, Gordon M, Roberts JM and Clurman BE. (1997). Genes Dev., 11, 1464 ± 1478. Shih HH, Tevosian SG and Yee AS. (1998). Mol. Cell. Biol., 18, 4732 ± 4743. Shin EK, Shin A, Paulding C, Schahausen B and Yee AS. (1995). Mol. Cell. Biol., 15, 2252 ± 2262. Shirodkar S, Ewen M, DeCaprio JA, Morgan J, Livingston DM and Chittenden T. (1992). Cell, 68, 157 ± 166. Shiyanov P, Bagchi S, Adami G, Kokontis J, Hay N, Arroyo M, Morozov A and Raychaudhuri P. (1996). Mol. Cell. Biol., 16, 737 ± 744. Singh P, Coe J and Hong W. (1995). Nature, 374, 562 ± 565. Skapek SX, Rhee J, Spicer DB and Lassar AB. (1995). Science, 267, 1022 ± 1024. Slansky JE and Farnham PJ. (1996). Curr. Top. Microbiol. Immunol., 208, 1 ± 30. Slansky JE, Li Y, Kaelin WG and Farnham PJ. (1993). Mol. Cell. Biol., 13, 1610 ± 1618. Smith EJ, Leone G, Degregori J, Jakoi L and Nevins JR. (1996). Mol. Cell. Biol., 16, 6965 ± 6976. Smith EJ, Leone G and Nevins JR. (1998). Cell Growth Dier., 9, 297 ± 303. Sterner JM, Dew-Knight S, Musahl C, Kornbluth S and Horowitz JM. (1998). Mol. Cell. Biol., 18, 2748 ± 2757. Strober BE, Dunaief JL, Guha and Go SP. (1996). Mol. Cell. Biol., 16, 1576 ± 1583. Stubdal H, Zalvide J, Campbell KS, Schweitzer C, Roberts TM and Decaprio JA. (1997). Mol. Cell. Biol., 17, 4979 ± 4990. Suzuki-Takahashi I, Kitagawa M, Saijo M, Higashi H, Ogino H, Matsumoto H, Taya Y, Nishimura S and Okuyama A. (1995). Oncogene, 10, 1691 ± 1698. Symonds H, Krall L, Remington L, Saenz RM, Lowe S, Jacks T and Van DT. (1994). Cell, 78, 703 ± 711. Tam SW, Theodoras AM, Shay JW, Draetta GF and Pagano M. (1994). Oncogene, 9, 2663 ± 2674. Tevosian SG, Shih HH, Mendelson KG, Sheppard KA, Paulson KE and Yee AS. (1997). Genes Dev., 11, 383 ± 396. Tommasi S and Pfeifer GP. (1995). Mol. Cell. Biol., 15, 6901 ± 6913.
Trimarchi JM, Fairchild B, Verona R, Moberg K, Andon N and Lees JA. (1998). Proc. Natl. Acad. Sci. USA, 95, 2850 ± 2855. Tsai KY, Hu Y, Macleod KF, Crowley D, Yamasaki L and Jacks T. (1998). Mol. Cell., 2, 293 ± 304. Vairo G, Livingston DM and Ginsberg D. (1995). Genes Dev., 9, 869 ± 881. Voit R, Schafer K and Grummt I. (1997). Mol. Cell. Biol., 17, 4230 ± 4237. Wang J, Huang Q, Tang W and Nadal-Ginard B. (1996). J. Cell. Biochem., 62, 405 ± 410. Weinberg RA. (1995). Cell, 81, 323 ± 330. White RJ. (1997). Trends Biochem Sci., 22, 77 ± 80. White RJ, Trouche D, Martin K, Jackson SP and Kouzarides T. (1996). Nature, 382, 88 ± 90. Wiggan O, Taniguchi-Sidle A and Hamel PA. (1998). Oncogene, 16, 227 ± 236. Williams BO, Schmitt EM, Remington L, Bronson RT, Albert DM, Weinberg RA and Jacks T. (1994). EMBO J., 13, 4251 ± 4259. Won KA and Reed SI. (1996). EMBO J., 15, 4182 ± 4193. Woo MS, Sanchez I and Dynlacht BD. (1997). Mol. Cell. Biol., 17, 3566 ± 3579. Wu X and Levine AJ. (1994). Proc. Natl. Acad. Sci. USA, 91, 3602 ± 3606. Xiao ZX, Ginsberg D, Ewen M and Livingston DM. (1996). Proc. Natl. Acad. Sci. USA, 93, 4633 ± 4637. Yamasaki L, Bronson R, Williams BO, Dyson NJ, Harlow E and Jacks T. (1998). Nat. Genet., 18, 360 ± 364. Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E and Dyson NJ. (1996). Cell, 85, 537 ± 548. Yan Z, DeGregori J, Shohet R, Leone G, Stillman B, Nevins JR and Williams RS. (1998). Proc. Natl. Acad. Sci. USA, 95, 3603 ± 3608. Yee AS, Shih HH and Tevosian SG. (1998). Front. Biosci., 3, d532 ± 547. Zacksenhaus E, Jiang Z, Chung D, Marth JD, Phillips RA and Gallie BL. (1996). Genes Dev., 10, 3051 ± 3064. Zalvide J and DeCaprio JA. (1995). Mol. Cell. Biol., 15, 5800 ± 5810. Zarkowska T and Mittnacht S. (1997). J. Biol. Chem., 272, 12738 ± 12746. Zarkowska T, U S, Harlow E and Mittnacht S. (1997). Oncogene, 14, 249 ± 254. Zerfass K, Schulze A, Spitkovsky D, Friedman V, Henglein B and Jansen-Durr P. (1995). J. Virol., 69, 6389 ± 6399. Zerfass-Thome K, Schulze A, Zwerschke W, Vogt B, Helin K, Bartek J, Henglein B and Jansen-Durr P. (1997). Mol. Cell. Biol., 17, 407 ± 415. Zhu L, Enders G, Lees JA, Beijersbergen RL, Bernards R and Harlow E. (1995a). EMBO J., 14, 1904 ± 1913. Zhu L, Harlow E and Dynlacht BD. (1995b). Genes Dev., 9, 1740 ± 1752. Zhu L, van den Heuvel S, Helin K, Fattaey A, Ewen M, Livingston D, Dyson N and Harlow E. (1993). Genes Dev., 7, 1111 ± 1125. Zhu L, Zhu L, Xie E and Chang LS. (1995c). Mol. Cell. Biol., 15, 3552 ± 3562. Zwicker J, Gross C, Lucibello FC, Truss M, Ehlert F, Engeland K and Muller R. (1995a). Nucleic Acids Res., 23, 3822 ± 3830. Zwicker J, Lucibello FC, Wolfraim LA, Gross C, Truss M, Engeland K and Muller R. (1995b). EMBO J., 14, 4514 ± 4522.
3383
