A key component of the machinery that regulates cell cycle entry and progression in mammalian cells is the retinoblastoma protein (Rb), which functions as a barrier to inappropriate cell cycle progression. The cyclin-dependent kinase (CDK) pathway controlling Rb is deregulated in most human tumors through deregulated expression of cyclins, inactivation of CDK inhibitors such as p16Ink4a or mutation of Rb itself (Sears and Nevins, 2002; Sherr and Roberts, 1999). The poster highlights some of the pathways that regulate Rb activity, as well as the mechanisms by which Rb regulates proliferation, apoptosis and differentiation.
Rb is a member of a gene family encoding structurally and functionally similar proteins, which include the Rb, p107 and p130 proteins (Stevaux and Dyson, 2002). Like Rb, p107 and p130 are regulated during the cell cycle by CDK phosphorylation, although there are clear differences at the levels of both function and expression. In addition, Rb family members associate with the cellular transcription factor E2F, negatively regulating E2F-dependent transcription. E2F activity plays crucial roles in cell cycle progression by regulating the transcription of genes involved in cell cycle regulation, DNA replication and mitosis (DeGregori, 2002; Trimarchi and Lees, 2002). E2F transcription factors form various heterodimers that are each composed of one E2F subunit and one DP subunit. E2F1, E2F2 and E2F3 family members predominantly associate with Rb, whereas E2F4 associates with Rb, p107 and p130. E2F5 appears to associate predominantly with p130. E2F6 and E2F7 appear to repress transcription via Rb-independent mechanisms. For simplicity, I use Rb here to refer generically to all three Rb family members.
Starting in late G1 phase of the cell cycle, Rb is heavily phosphorylated until mitosis. Hypophosphorylated Rb is the active form of Rb that negatively regulates E2F and cell cycle entry. The hyperphosphorylation of Rb during G1 progression is largely carried out by CDKs. Specifically, in mid-G1 phase, Rb is first phosphorylated by Cyclin-D-dependent CDKs (CDK4 or CDK6), which associate with one of three D-type cyclins. The D-type cyclin-CDKs are highly responsive to growth factor (GF) stimulation at several levels. These include synthesis of their subunits, association with inhibitory proteins such as cyclin kinase inhibitors (CKIs), assembly of the subunits, and cyclin stability (Sherr and Roberts, 1999). Both the transcription of the cyclin D1 gene and assembly of cyclin D1 with CDK4 or CDK6 depend on Ras activation. Furthermore, increased Myc activity in early to mid G1 phase promotes the transcription of the genes that encode cyclin D1, cyclin D2 and CDK4 (Sears and Nevins, 2002). In a quiescent cell, the kinase GSK3β phosphorylates cyclin D1, resulting in its cytoplasmic retention and degradation. Following GF receptor (GF-R) signaling via Ras and phosphoinositide 3-kinase (PI 3-kinase), the kinase AKT (also known as PKB) inhibits GSK3β activity, allowing the accumulation of cyclin D1 in the nucleus (Sherr and Roberts, 1999).
CKIs of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d) specifically associate with CDK4 and CDK6, blocking the kinase active site and preventing association with cyclins. By contrast, CKIs of the CIP/KIP family (p21Cip1, p27Kip1 and p57Kip2) associate with and potently inhibit cyclin-E- and cyclin-A-dependent CDKs. Association of the cyclin-D–CDK4/6 complex with p21Cip1 and p27Kip1 following GF activation is required for cyclin-E–CDK2 activation, sequestering these CKIs away from cyclin-E–CDK2. Interestingly, p21Cip1 and p27Kip1 are also required for the assembly and nuclear accumulation of cyclin-D–CDK complexes (Sherr and Roberts, 1999). The CKIs are controlled at multiple levels by extra- and intra-cellular mediated signaling. TGFβ signaling, which generally inhibits cell cycle progression, increases the transcription of the p15-encoding Ink4b and p21-encoding Cip1 genes. Similarly, cellular stress or DNA damage can activate p53, which promotes the transcription of Cip1, leading to CDK inhibition, Rb activation (reduced phosphorylation) and cell cycle arrest. Furthermore, the Ras-MAPK (mitogen activated protein kinase) pathway may mediate increased p16Ink4a expression during senescence induction. By contrast, AKT activation by GF signaling results in increased cytoplasmic localization of p21Cip1 and p27Kip1 (Zhou and Hung, 2002). Finally, Myc promotes the transcription of Cul1, which encodes a component of the SCF ubiquitin ligase that promotes the degradation of p27Kip1 protein, contributing to p27Kip1 downregulation during G1.
The sequential and combined phosphorylation of Rb by cyclin-D- and cyclin-E-dependent CDKs contributes to inactivation of Rb (Sherr and Roberts, 1999; Stevaux and Dyson, 2002). Cyclin-A-dependent CDK activity might also contribute to the maintenance of Rb inactivation during S-G2 phase progression. The dephosphorylation of Rb is also important to reactivate Rb (although Rb might never be totally unphosphorylated), either following mitosis or in response to GF withdrawal, and appears to be mediated by the combined action of phosphatases together with CDK inactivation. Rb is also regulated following apoptotic stimulation by caspase-mediated cleavage, which leads to Rb degradation (Chau and Wang, 2003). Indeed, Rb cleavage has been shown to be critical for TNFα-induced apoptosis. Rb can negatively regulate apoptosis by associating with and inhibiting of the Abl and JNK kinases, as well as by inhibiting the expression of proapoptotic E2F target genes such as those encoding the p53 family member p73, APAF1 and some caspases (Chau and Wang, 2003). Similar roles for p107 and p130 in negatively regulating apoptosis have not been demonstrated.
GF-dependent activation of cyclin–CDK-mediated Rb phosphorylation and E2F activation are necessary (but probably not sufficient) for cell cycle entry and progression. Association of Rb with E2F masks the transcriptional activation domain of E2F. Importantly, Rb also functions as an active transcriptional repressor by recruiting various cofactors, many of which are involved in remodeling chromatin (Stevaux and Dyson, 2002). Rb-E2F recruits histone deacetylases (HDACs) to E2F target promoters; these remove acetyl groups from histone proteins at the promoter, contributing to a closed chromatin state and transcriptional repression. In quiescent cells, E2F-binding sites at E2F-regulated promoters are occupied by E2F4, p130 and HDAC, and this coincides with reduced histone H3 acetylation and decreased gene expression. Following GF stimulation, in late G1 phase these same E2F-binding sites become occupied by E2F1, E2F2 and E2F3, and this coincides with increased histone H3 acetylation and transcription, which is consistent with the ability of E2Fs to associate with histone acetyl transferases (HATs). Rb family members also appear to mediate gene repression via the recruitment of Polycomb group (PcG) proteins and components of the Swi/Snf chromatin-remodeling complexes (Stevaux and Dyson, 2002).
Notably, in several studies, investigators have shown that Rb (as opposed to p107 and p130) is not present at E2F-regulated promoters in either quiescent or GF activated cells. By contrast, Rb is found associated with these promoters in senescent cells, which results in stable repression of E2F-dependent transcription via the recruitment of the SUV39 histone methytransferase (HMT). SUV39 methylation of histone H3 results in the recruitment of heterochromatin protein 1 (HP1), which promotes the formation of heterochromatin. SUV39-mediated methylation of histone H3 might require prior deacetylation of the same Lys9 residue on histone H3 by HDAC activities. Notice that the role of HDAC, HMT and chromatin-remodeling complex (such as Swi/Snf) activities in Rb-mediated repression during the cell cycle, quiescence or senescence have not been fully established. The complexes shown should therefore not be interpreted to reflect, for example, distinct roles for HDACs and HMTs in Rb-mediated gene repression during quiescence and senescence, respectively.
Rb has also been shown to promote the transcription of differentiation mediators – for example, following its recruitment by the CBFA1 transcription factor to promoters of genes encoding osteogenic factors and by C/EBP proteins to promoters of genes encoding adipogenic factors (Thomas et al., 2003). Thus, in contrast to p107 and p130, Rb may play a more specific role in regulating gene expression during differentiation and senescence. Finally, Rb and E2Fs have been shown to associate near replication origins in both mammalian and Drosophila model systems, which suggests that they might have direct roles in regulating DNA replication (Stevaux and Dyson, 2002).
- © The Company of Biologists Limited 2004