The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis.
The first appearance of G1 during Drosophila embryogenesis, at cell cycle 17, is accompanied by the down-regulation of E2F-dependent transcription. Mutant alleles of rbf were generated and analyzed to determine the role of RBF in this process. Embryos lacking both maternal and zygotic RBF products show constitutive expression of PCNA and RNR2, two E2F-regulated genes, indicating that RBF is required for their transcriptional repression. Despite the ubiquitous expression of E2F target genes, most epidermal cells enter G1 normally. Rather than pausing in G1 until the appropriate time for cell cycle progression, many of these cells enter an ectopic S-phase. These results indicate that the repression of E2F target genes by RBF is necessary for the maintenance but not the initiation of a G1 phase. The phenotype of RBF-deficient embryos suggests that rbf has a function that is complementary to the roles of dacapo and fizzy-related in the introduction of G1 during Drosophila embryogenesis. (+info
Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins.
Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells. (+info
Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.
The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo. (+info
Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity.
The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells. (+info
Analysis of the degradation function of Mdm2.
Degradation of the p53 tumor suppressor protein has been shown to be regulated by Mdm2. In this study, we identify regions of Mdm2 that are not required for p53 binding but are essential for degradation. Mdm2 mutants lacking these regions function in a dominant negative fashion, stabilizing endogenous p53 in cells by interfering with the degradative function of the endogenous Mdm2. p53 protein stabilized in this way does not strongly enhance the expression of p21(Waf1/Cip1), the product of a p53-responsive gene, supporting the model in which binding of Mdm2 to the NH2-terminal domain of p53 inhibits interaction with other components of the basal transcriptional machinery. Interestingly, COOH-terminal truncations of Mdm2 that retain p53 binding but fail to mediate its degradation are also stabilized themselves. Because Mdm2, like p53, is normally an unstable protein that is degraded through the proteasome, this result suggests a direct link between the regulation of Mdm2 and p53 stability. (+info
Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases.
The E2F transcription factor plays a major role in cell cycle regulation, differentiation and apoptosis, but it is not clear how it is regulated by non-mitogenic signaling cascades. Here we report that two kinases involved in signal transduction have opposite effects on E2F function: the stress-induced kinase JNK1 inhibits E2F1 activity whereas the related p38 kinase reverses Rb-mediated repression of E2F1. JNK1 phosphorylates E2F1 in vitro, and co-transfection of JNK1 reduces the DNA binding activity of E2F1; treatment of cells with TNFalpha had a similar effect. Fas stimulation of Jurkat cells is known to induce p38 kinase and we find a pronounced increase in Rb phosphorylation within 30 min of Fas stimulation. Phosphorylation of Rb correlated with a dissociation of E2F and increased transcriptional activity. The inactivation of Rb by Fas was blocked by SB203580, a p38-specific inhibitor, as well as a dominant-negative p38 construct; cyclin-dependent kinase (cdk) inhibitors as well as dominant-negative cdks had no effect. These results suggest that Fas-mediated inactivation of Rb is mediated via the p38 kinase, independent of cdks. The Rb/E2F-mediated cell cycle regulatory pathway appears to be a normal target for non-mitogenic signaling cascades and could be involved in mediating the cellular effects of such signals. (+info
Regulation of endogenous E2F1 stability by the retinoblastoma family proteins.
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 increased markedly. E2F1 stability also was 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 disrupted 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. (+info
RB regulates the stability and the apoptotic function of p53 via MDM2.
The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53. (+info