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1.  Distinct activities of the anaphase-promoting complex/cyclosome (APC/C) in mouse embryonic cells 
Cell Cycle  2012;11(5):846-855.
The first differentiation event in mammalian development gives rise to the blastocyst, consisting of two cell lineages that have also segregated in how the cell cycle is structured. Pluripotent cells of the inner cell mass divide mitotically to retain a diploid DNA content, but the outer trophoblast cells can amplify their genomes more than 500-fold by undergoing multiple rounds of DNA replication, completely bypassing mitosis. Central to this striking divergence in cell cycle control is the E3 ubiquitin-ligase activity of the anaphase-promoting complex or cyclosome (APC/C). Extended suppression of APC/C activity during interphase of mouse pluripotent cells promotes rapid cell cycle progression by allowing stabilization of cyclins, whereas unopposed APC/C activity during S phase of mouse trophoblast cells triggers proteasomal-mediated degradation of geminin and giant cell formation. While differential APC/C activity might govern the atypical cell cycles observed in pre-implantation mouse embryos, geminin is a critical APC/C substrate that: (1) escapes degradation in pluripotent cells to maintain expression of Oct4, Sox2 and Nanog and (2) mediates specification and endoreduplication when targeted for ectopic destruction in trophoblast. Thus, in contrast to trophoblast giant cells that lack geminin, geminin is preserved in both mouse pluripotent cells and non-endoreduplicating human cytotrophoblast cells.
doi:10.4161/cc.11.5.19251
PMCID: PMC3323792  PMID: 22333576
APC/C; geminin; Emi1; cell cycle; pluripotency; trophoblast; endoreduplication; DNA damage
2.  MDM2 promotes SUMO-2/3 modification of p53 to modulate transcriptional activity 
Cell Cycle  2011;10(18):3176-3188.
The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.
doi:10.4161/cc.10.18.17436
PMCID: PMC3218624  PMID: 21900752
p53; SUMO-2/3; sumoylation; MDM2; ARF; L11
3.  A High-Throughput Computational Framework for Identifying Significant Copy Number Aberrations from Array Comparative Genomic Hybridisation Data 
Advances in Bioinformatics  2012;2012:876976.
Reliable identification of copy number aberrations (CNA) from comparative genomic hybridization data would be improved by the availability of a generalised method for processing large datasets. To this end, we developed swatCGH, a data analysis framework and region detection heuristic for computational grids. swatCGH analyses sequentially displaced (sliding) windows of neighbouring probes and applies adaptive thresholds of varying stringency to identify the 10% of each chromosome that contains the most frequently occurring CNAs. We used the method to analyse a published dataset, comparing data preprocessed using four different DNA segmentation algorithms, and two methods for prioritising the detected CNAs. The consolidated list of the most commonly detected aberrations confirmed the value of swatCGH as a simplified high-throughput method for identifying biologically significant CNA regions of interest.
doi:10.1155/2012/876976
PMCID: PMC3449101  PMID: 23008709
4.  Geminin Escapes Degradation in G1 of Mouse Pluripotent Cells and Mediates the Expression of Oct4, Sox2, and Nanog 
Current Biology  2011;21(8):692-699.
Summary
Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells [1, 2]. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast [3, 4]. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1 [1, 2, 5]. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?
Highlights
► Geminin is essential for pluripotency of mouse EC and ES cells ► Accordingly, mouse EC and ES cells retain geminin in G1 ► By activating APC/CCdh1, Emi1 depletion phenocopies depletion of geminin ► Geminin sustains core pluripotency factors by antagonizing chromatin remodeler Brg1
doi:10.1016/j.cub.2011.03.026
PMCID: PMC3083515  PMID: 21497086
5.  A role for Numb in p53 stabilization 
Genome Biology  2008;9(5):221.
Loss of Numb could enable breast cancer cells to bypass the p53 tumor suppressor response
The cell-fate determinant Numb has recently been shown to help activate the tumor suppressor protein p53. Loss of Numb in breast cancers would result, therefore, in both the activation of the potential oncogene Notch and the diminution of tumor suppression by p53.
doi:10.1186/gb-2008-9-5-221
PMCID: PMC2441455  PMID: 18492217
6.  Endosomal Dynamics of Met Determine Signaling Output 
Molecular Biology of the Cell  2003;14(4):1346-1354.
Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (Hammond et al., 2001). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5–10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.
doi:10.1091/mbc.E02-09-0578
PMCID: PMC153105  PMID: 12686592

Results 1-6 (6)