RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2

Expression of a dominant negative 20-kDa isoform of CCAAT/enhancer-binding protein (C/EBPbeta), LIP, is increased in proliferating livers and in tumor cells. Two RNA-binding proteins, CUGBP1 and calreticulin, have been implicated in the translational regulation of C/EBPbeta. In this paper, we present evidence showing several critical steps by which liver increases translation of LIP after partial hepatectomy. At early stages after partial hepatectomy, liver activates CUGBP1 by a hyperphosphorylation. The activated CUGBP1 binds to the 5' region of C/EBPbeta mRNA and replaces calreticulin, which partially represses translation of C/EBPbeta in quiescent livers. The hyperphosphorylated CUGBP1 also interacts with the alpha and beta subunits of initiation factor eIF2. Our data demonstrate that the interaction of CUGBP1 with the eIF2alpha enhances the association of CUGBP1 with ribosomes and correlates with increased translation of LIP in the liver after partial hepatectomy. Our data support the hypothesis that CUGBP1 increases translation of LIP by the interaction with the eIF2alpha subunit. This facilitates subsequent recruitment of larger numbers of ribosomes to initiate translation of LIP.     

Age-specific CUGBP1-eIF2 complex increases translation of CCAAT/enhancer-binding protein beta in old liver

The RNA-binding protein CUGBP1 regulates translation of proteins in a variety of biological processes. In this study, we show that aging liver increases CUGBP1 translational activities by induction of a high molecular weight protein-protein complex of CUGBP1. The complex contains CUGBP1, subunits alpha, beta, and gamma of the initiation translation factor eIF2, and four proteins of the endoplasmic reticulum, eR90, CRT, eR60, and Grp78. The induction of the CUGBP1-eIF2 complex in old livers is associated with the elevation of protein levels of CUGBP1 and with the hyper-phosphorylation of CUGBP1 by a cyclin D3-cdk4 kinase, activity of which is increased with age. We have examined the role of the elevation of CUGBP1 and the role of cyclin D3-cdk4-mediated phosphorylation of CUGBP1 in the formation of the CUGBP1-eIF2 complex by using CUGBP1 transgenic mice and young animals expressing high levels of cyclin D3 after injection with cyclin D3 plasmid. These studies showed that both the increased levels of CUGBP1 and cdk4-mediated hyper-phosphorylation of CUGBP1 are involved in the age-associated induction of the CUGBP1-eIF2 complex. The CUGBP1-eIF2 complex is bound to C/EBPbeta mRNA in the liver of old animals, and this binding correlates with the increased amounts of liver-enriched activator protein and liver-enriched inhibitory protein. Consistent with these observations, the purified CUGBP1-eIF2 complex binds to the 5' region of C/EBPbeta mRNA and significantly increases translation of the three isoforms of C/EBPbeta in a cell-free translation system, in cultured cells, and in the liver. Thus, these studies demonstrated that age-mediated induction of the CUGBP1-eIF2 complex changes translation of C/EBPbeta in old livers.     

C/EBPalpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPalpha. We have observed that C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.     

Dephosphorylated C/EBPalpha accelerates cell proliferation through sequestering retinoblastoma protein

CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been previously considered a strong inhibitor of cell proliferation which uses multiple pathways to cause growth arrest. In this paper, we describe a new function of C/EBPalpha, which is an acceleration of cell proliferation. This new function of C/EBPalpha is created in proliferating livers by protein phosphatase 2A-mediated dephosphorylation of C/EBPalpha at Ser193. The Ser193-dephosphorylated C/EBPalpha interacts with retinoblastoma protein (Rb) independently on E2Fs and sequesters Rb, leading to a reduction of E2F-Rb repressors and to acceleration of proliferation. This new function of C/EBPalpha requires Rb, since the dephosphorylated C/EBPalpha does not promote proliferation in Rb-negative cells. We also show that a balance of Rb and Ser193-dephosphorylated C/EBPalpha determines if the cells are growth arrested or have an increased rate of proliferation. Consistently with these findings, a significant portion of Rb is sequestered into Rb-C/EBPalpha complexes in proliferating livers, and E2F-Rb complexes are not detectable in these livers. Our data demonstrate a new pathway by which the phosphorylation-dependent switch of biological functions of C/EBPalpha promotes liver proliferation.     

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.