Interactive Fly, Drosophila

Interaction of Retinoblastoma proteins with E2Fs

E2F-1 (Drosophila homolog: E2F) plays a crucial role in the regulation of cell-cycle progression at the G1-S transition. When overproduced, it is both an oncoprotein and a potent inducer of apoptosis; therefore, its transcriptional activity is subject to multiple controls. Among these multiple controls are binding by the retinoblastoma gene product (pRb), activation by cdk3, and S-phase-dependent down-regulation of DNA-binding capacity by cyclin A-dependent kinase. E2F-1 is actively degraded by the ubiquitin-proteasome pathway. Efficient degradation depends on the availability of selected E2F-1 sequences. Unphosphorylated pRb stabilizes E2F-1, protecting it from in vivo degradation. pRb-mediated stabilization is not an indirect consequence of G1 arrest, but rather depends upon the ability of pRb to interact physically with E2F-1. Thus, in addition to binding E2F-1 and transforming it into a transcriptional repressor, pRb has another function: protection of E2F-1 from efficient degradation during a period when pRb/E2F complex formation is essential to regulating the cell cycle. In addition, there may be a specific mechanism for limiting free E2F-1 levels. The failure of such a mechanism could compromise cell survival and/or homeostasis (Hofmann, 1996).

Certain E2F transcription factor species play a pivotal role in regulating cell-cycle progression. The activity of E2F1, a protein with neoplastic transforming activity when unregulated, is tightly controlled at the transcriptional level during G0 exit. In addition, during this interval, the stability of endogenous E2F1 protein increases markedly. E2F1 stability also is dynamically regulated during myogenic differentiation and in response to gamma irradiation. One or more retinoblastoma family proteins likely participate in the stability process, because simian virus 40 T antigen disruptes E2F1 stability regulation during G1 exit in a manner dependent on its ability to bind to pocket proteins. Thus, endogenous E2F1 function is regulated by both transcriptional and posttranscriptional control mechanisms (Martelli, 1999).

Rb inhibits progression from G1 to S phase of the cell cycle. It associates with a number of cellular proteins; however, the nature of these interactions and their relative significance in cell cycle regulation are still unclear. Evidence is presented that Rb must normally interact with the E2F family of transcription factors to arrest cells in G1, and that this arrest results from active transcriptional repression by the Rb-E2F complex, not from inactivation of E2F. Thus, a major role of E2F in cell cycle regulation is assembly of this repressor complex. Active repression by Rb-E2F mediates the G1 arrest triggered by both TGFbeta and p16INK4a and also mediates contact inhibition (Zhang, 1999).

Electrophoretic mobility shift assays were used to analyse the pattern of E2F transcription factor complexes containing pRB and related 'pocket' proteins associated with changes in growth of monkey CV-1 cells. Little change is noted in pRB/E2F complexes following growth arrest or serum stimulation. Serum starvation induces the formation of a novel slowly-migrating p130/E2F complex, termed C7, which is comparable to one reported previously in terminally differentiated C2C12 mouse cells and thought to contain one or more additional unidentified proteins. After serum stimulation, C7 complex disappears in S-phase but returns during mitosis. A major E2F complex containing p107 appears during S-phase but is undetectable at other times. It appears likely that regulation of pRB, p107 and p130 occurs by several mechanisms:

Phosphorylation has previously been proposed to be involved. All pRB-related proteins are found to be highly phosphorylated during S-phase when E2F activity is highest, and hypophosphorylated in arrested cells when E2F activity is low.
The absolute levels of some members of the RB family vary during the cell cycle. Levels of pRB protein and pRB/E2F complexes change little; however, the amounts of both p107/E2F and the growth arrest form, p130/E2F, vary according to the overall levels of p107 and p130 proteins.
Formation of the novel C7 complex in serum arrested cells suggests a third regulatory mechanism mediated by interactions with additional proteins.

Binding of such proteins with p130/E2F occurs via a site in the 'pocket' similar to that utilized by adenovirus E1A proteins. Such proteins could function as additional regulators of E2F-driven transcription (Corbeil, 1997).

Free E2F-1 and E2F-4 transcription factors are unstable; their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. E2F-1 interaction with pRb and E2F-4 interaction with p107 or p130 protects these E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins (Hateboer, 1996).

The E2F transcription factor couples the coordinate expression of cell cycle proteins to their appropriate transition points. Its activity is controlled by the cell cycle regulators pRB, p107, and p130. These bind to E2F at defined but distinct stages of the cell cycle. Using specific antisera, the DP and E2F components of each of these species has been identified. Although present at very different levels, DP-1 and DP-2 are evenly distributed among each of these complexes. In contrast, the individual E2Fs have distinctly different binding profiles. Consistent with previous studies, E2F-1, E2F-2, and E2F-3 bind specifically to the retinoblastoma protein. In each case, their expression and DNA binding activity are restricted to post-G1/S fractions. Surprisingly, E2F-1 and E2F-3 make unequal contributions to the pRB-associated and free E2F activity, suggesting that these proteins perform different cell cycle functions. Most significantly, this study shows E2F-4 accounts for the vast majority of the endogenous E2F activity. In arrested cells, E2F-4 is sequestered by the p130 protein. However, as the cells pass the G1-to-S transition, the levels of pRB and p107 increase and E2F-4 now associates with both of these regulators. Despite this, a considerable amount of E2F-4 exists as free E2F. In G1 cells, this accounts for almost all of the free activity. Once the cells enter S phase, free E2F is composed of an equal mixture of E2F-4 and E2F-1 (Moberg, 1996).

Mice mutant for the Rb tumor suppressor gene die in mid-gestation with defects in erythropoiesis, cell cycle control, and apoptosis. Embryos mutant for both Rb and its downstream target E2f-1 demonstrate significant suppression of apoptosis and S phase entry in certain tissues as compared to Rb mutants, implicating E2f-1 as a critical mediator of these effects. Up-regulation of the p53 pathway, required for cell death in these cells in Rb mutants, is also suppressed in the Rb/E2f-1 double mutants. However, double mutants have defects in cell cycle regulation and apoptosis in some tissues and die at approximately E17.0 with anemia and defective skeletal muscle and lung development, demonstrating that E2F-1 regulation is not the sole function of pRB in development (Tsai, 1998).

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (1) the A/B pocket, which binds proteins with the LXCXE motif; (2) the C pocket, which binds the c-Abl tyrosine kinase (see Drosophila Abl oncogene); and (3) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). The E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB (Knudsen, 1997).

Cyclin E is critical for the advance of cells through the G1 phase of their growth cycle. Transcription of the cyclin E gene is known to be cell cycle-dependent. mRNA levels of cyclin E are regulated positively by mitogens and negatively by TGF-beta. Much circumstantial evidence implicates both E2F transcription factors and the retinoblastoma protein (pRB) in the control of cyclin E expression. However, the molecular basis of this control has remained unclear. There are several putative E2F binding sites within the cyclin E promoter sequence. Cell cycle regulation of cyclin E transcription is mediated by E2F binding sites present in the promoter. The activity of this promoter can be regulated negatively by pRB. These results suggest the operation of a positive-feedback loop in late G1 that functions to ensure continued cyclin E expression and pRB inactivation (Geng, 1996).

Histone gene expression is restricted to the S phase of the cell cycle. Control is mediated by a complex network of sequence-specific DNA-binding factors and protein-protein interactions in response to cell cycle progression. To further investigate the regulatory functions that are associated at the transcriptional level, the regulation of a replication-dependent human H2A.1-H2B.2 gene pair was analyzed. Transcription factor E2F binds specifically to an E2F recognition motif in the H2A.1 promoter region. Activation of the H2A.1 promoter by E2F-1 was shown by use of luciferase reporter constructs of the intergenic promoter region. Overexpression of the human retinoblastoma suppressor gene product RB suppresses E2F-1 mediated transcriptional activation, indicating an E2F-dependent regulation of promoter activity during the G1-to-S-phase transition. The activity of the H2A.1 promoter is also downregulated by overexpression of the RB-related p107, a protein that has been detected in S-phase-specific protein complexes of cyclin A, E2F, and cdk2. In synchronized HeLa cells, expression of luciferase activity is induced at the beginning of DNA synthesis and is dependent on the presence of an E2F-binding site in the H2A.1 promoter. Together with the finding that E2F-binding motifs are highly conserved in H2A promoters of other species, these results suggest that E2F plays an important role in the coordinate regulation of S-phase-specific histone gene expression (Oswald, 1996).

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. The transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. E2F-4 accounts for the majority of endogenous E2F species. The subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed (Verona, 1997).

Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. Mutations have been constructed that disrupt C pocket activity. When overproduced, the RB C-terminal fragment does not induce terminal growth arrest but can inhibit G1/S progression, and this activity is abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduce but do not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolish RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression (Whitaker, 1998).

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (1) the A/B pocket, which binds proteins with the LXCXE motif; (2) the C pocket, which binds the c-Abl tyrosine kinase, and (3) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). The E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Additionally, phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind the LXCXE proteins, c-Abl, and E2F, even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB (Knudsen, 1997).

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. A mutant mouse model was generated to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 has no detectable effect on either cell cycle arrest or proliferation. However, E2F4 is essential for normal development. E2f4-/- mice die of an increased susceptibility to opportunistic infections that appears to result from craniofacial defects. They also display a variety of erythroid abnormalities that arise from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor (Humbert, 2000).

E2F4 accounts for the majority of endogenous E2F DNA binding activity and is thought to play a key role in mediating the transcriptional repressive properties of pRB, p107, and p130. Consequently, comparison of the phenotypes of E2f4 and Rb, p107 and p130 mutant mice provides considerable insight into the biological roles of the E2F•pocket protein complexes. Mice deficient for p107 and p130 have a defect in long bone development that results from the failure of chondrocytes to arrest and differentiate at the correct developmental stage. In addition, p107-/-:p130-/- mutant MEFs enter S phase prematurely, and this correlates with the dramatic deregulation of many E2F-responsive genes. These data support the notion that p107•E2F and p130•E2F complexes play a key role in mediating the repression of E2F-responsive genes during G0/G1. However, loss of E2F-4, and therefore the vast majority of the p107•E2F and p130•E2F complexes, does not cause any defect in long bone formation or cell cycle regulation and it has little or no effect on the expression of E2F-responsive genes. Thus, loss of E2F4 is not equivalent to loss of p107 and p130. These findings support two alternative models of p107/p130 action. First, correct cell cycle regulation could be dependent upon the formation of transcriptionally repressive p107•E2F and p130•E2F complexes but E2F5 is sufficient to mediate this effect in the absence of E2F4. Alternatively, loss of p107 and p130 could lead to the deregulation of E2F-responsive genes and thereby S phase entry through the inappropriate release of 'free', transcriptionally active E2F4 and E2F5 complexes. This would be entirely analogous to the finding that ectopic S phase entry and aberrant apoptosis in Rb-deficient embryos or tumors is largely due to the inappropriate activation of E2F1, a pRB-specific E2F. However, it is inconsistent with the current belief that, at least in normal cells, E2F4 and E2F5 make little or no contribution to the activation of E2F-responsive genes (Humbert, 2000 and references therein).

In contrast to p107 and p130, significant similarities were found in the roles of E2F4 and pRB in vivo. pRb-deficient mice die at E13.5 from ineffective erythropoiesis. This phenotype is complex and results from defects in both RBCs and hepatocytes. However, there is evidence to support the presence of a cell-autonomous component to the erythroid defect. (1) Embryos with high Rb-/- ES cell contribution display abnormal nucleated erythrocytes peaking at E16.5 of development. (2) pRB-deficient erythroid progenitor cells can repopulate the bone marrow and periphery of wild-type adoptive transfer animals but there is a high frequency of nucleated erythrocytes, and these animals develop anemia within a year. E2F4 also plays a critical role in normal erythroid maturation in a cell intrinsic manner. Taken together, these data suggest that E2F4 makes a major contribution to the regulation of erythropoietic system development by the RB tumor suppressor (Humbert, 2000 and references therein).

The interaction between pRB and E2F is critical for control of the cell cycle and apoptosis. pRB contains two distinct E2F binding sites. The previously identified E2F binding site on pRB is necessary for stable association with E2Fs on DNA. A second E2F interaction site is located entirely within the C-terminal domain of pRB and is specific for E2F1. E2F1/pRB complexes formed through this site have low affinity for DNA, but the interaction is sufficient for pRB to regulate E2F1-induced apoptosis, and E2F1 loses the ability to interact with this site following DNA damage. These results show that pRB interacts with individual E2F proteins in different ways and suggest that pRB's regulation of E2F1-induced apoptosis is physically separable from its transcriptional control of other E2F proteins (Dick, 2003).

Table of contents

Retinoblastoma-family protein: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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