Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

Cdc6, which alters chromatin ultrastructure to allow DNA replication in muscle stem cells transitioning out of quiescence, is identified as a target of the MyoD transcription factor

Expression of centromere protein F (CENP-F) associated with higher FDG uptake on PET/CT, detected by cDNA microarray, predicts high-risk patients with primary breast cancer

<p>Abstract</p> <p>Background</p> <p>Higher standardized uptake value (SUV) detected by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) correlates with proliferation of primary breast cancer. The purpose of this study is to identify specific molecules upregulated in primary breast cancers with a high SUV and to examine their clinical significance.</p> <p>Methods</p> <p>We compared mRNA expression profiles between 14 tumors with low SUVs and 24 tumors with high SUVs by cDNA microarray. We identified centromere protein F (CENP-F) and CDC6 were upregulated in tumors with high SUVs. RT-PCR and immunohistochemical analyses were performed to validate these data. Clinical implication of CENP-F and CDC6 was examined for 253 archival breast cancers by the tissue microarray.</p> <p>Results</p> <p>The relative ratios of CENP-F and CDC6 expression levels to β-actin were confirmed to be significantly higher in high SUV tumors than in low SUV tumors (<it>p </it>= 0.027 and 0.025, respectively) by RT-PCR. In immunohistochemical analysis of 47 node-negative tumors, the CENP-F expression was significantly higher in the high SUV tumors (74%) than the low SUV tumors (45%) (<it>p </it>= 0.04), but membranous and cytoplasmic CDC6 expressions did not significantly differ between both groups (<it>p </it>= 0.9 each). By the tissue microarray, CENP-F (HR = 2.94) as well as tumor size (HR = 4.49), nodal positivity (HR = 4.1), and Ki67 (HR = 2.05) showed independent impact on the patients' prognosis.</p> <p>Conclusion</p> <p>High CENP-F expression, correlated with high SUV, was the prognostic indicators of primary breast cancer. Tumoral SUV levels may serve as a pretherapeutic indicator of aggressiveness of breast cancer.</p

The Human TPR Protein TTC4 Is a Putative Hsp90 Co-Chaperone Which Interacts with CDC6 and Shows Alterations in Transformed Cells

BACKGROUND: The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein. CONCLUSIONS/SIGNIFICANCE: Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells

Cdk2 Is Required for p53-Independent G2/M Checkpoint Control

The activation of phase-specific cyclin-dependent kinases (Cdks) is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G2/M checkpoint activation

Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle

In eukaryotic cells, the cell-division cycle (CDC)-6 protein is essential to promote the assembly of pre-replicative complexes in the early G1 phase of the cell cycle, a process requiring tight regulation to ensure that proper origin licensing occurs once per cell cycle. Here we show that, in late G1 and early S phase, CDC6 is found in a complex also containing Cyclin A, cyclin-dependent kinase (CDK)-2 and the acetyltransferase general control nonderepressible 5 (GCN5). GCN5 specifically acetylates CDC6 at three lysine residues flanking its cyclin-docking motif, and this modification is crucial for the subsequent phosphorylation of the protein by Cyclin A-CDKs at a specific residue close to the acetylation site. GCN5-mediated acetylation and site-specific phosphorylation of CDC6 are both necessary for the relocalization of the protein to the cell cytoplasm in the S phase, as well as to regulate its stability. This two-step, intramolecular regulatory program by sequential modification of CDC6 seems to be essential for proper S-phase progression

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment

Cdc6 Stability Is Regulated by the Huwe1 Ubiquitin Ligase after DNA Damage

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APCCdh1) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APCCdh1 ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage