G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

Cell cycle-related transformation of the E2F4-p130 repressor complex

During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.     

E2F/p107 and E2F/p130 complexes are regulated by C/EBPalpha in 3T3-L1 adipocytes.

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.     

Distinct cellular factors regulate the c-myb promoter through its E2F element

Most E2F-driven promoters are transiently activated around the G(1)/S transition. Although the promoter for the c-myb proto-oncogene harbors an E2F element, it is induced early in G(1) following entry into the cell cycle. Furthermore, this promoter remains active throughout subsequent cell cycles. Since E2F sites function as repressor elements during G(1) (due to the association of pRb with E2F factors), we investigated whether the E2F element in the c-myb promoter is regulated differently than E2F elements in promoters that are repressed during G(1). By gel shift analysis, the E2F element from the c-myb promoter was found to form a unique complex, referred to as E2Fmyb-sp, which was not observed with E2F elements from several other promoters. Antibodies to DP-1, E2F1 to -5, p107, or pRb failed to either supershift or block E2Fmyb-sp complex formation. Methylation interference experiments indicate that the DNA contact residues for the E2Fmyb-sp complex are distinct from but overlapping with residues required for the binding of E2F proteins. In addition to the identification of E2Fmyb-sp, we have found that SP-1 binds to the c-myb E2F element. Functional studies revealed that E2Fmyb-sp and/or SP-1 are required to achieve full activation of the c-myb promoter in different cell types and to maintain elevated expression of the c-myb promoter during G(1) in NIH 3T3 cells. These studies demonstrate that E2F elements can be regulated differently through the binding of unique sets of proteins.