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.     

Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression

The proto-oncogene c-myc is a key player in cell-cycle regulation and is deregulated in a broad range of human cancers and cell proliferation disorders. Here we reported that overexpression of c-myc in human embryonic lung fibroblasts (HEL) that have low endogenous c-myc enriched S phase cells with increased expression of cyclin D3, E, A, Cdk2, and Cdk4, and decreased expression of p21 and p27. To the opposite, using RNAi to downregulate c-myc expression in A549 cells that have high endogenous c-myc enriched G1 phase cells with decreased expression of cyclin D3, E, A, Cdk2, Cdk4, and increased expression of p21 and p27. We found that cyclin A expression was the most susceptive to changes in c-myc levels and essential in c-myc-modulated cell cycle pathway via co-transfection, however, cyclin D1 showed no change between treated and control groups in either HEL or A549 cells. Our results indicated that upregulation of c-myc expression promotes cell cycling in HEL cells, whereas downregulation of c-myc expression causes G1 phase arrest in A549 cells, and the c-myc-mediated cell-cycle regulation pathway was dependent on cyclin A and involved cyclin D3, E, Cdk2, Cdk4, p21, and p27, but not cyclin D1.     

A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB.     

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.     

Cdh1 regulates osteoblast function through an APC/C-independent modulation of Smurf1

The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell-cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell-cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here, we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an autoinhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets, including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell-cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.