Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.     

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.     

Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2)

The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.     

Structural basis for cell cycle checkpoint control by the BRCA1-CtIP complex

The breast and ovarian tumor suppressor BRCA1 has important functions in cell cycle checkpoint control and DNA repair. Two tandem BRCA1 C-terminal (BRCT) domains are essential for the tumor suppression activity of BRCA1 and interact in a phosphorylation-dependent manner with proteins involved in DNA damage-induced checkpoint control, including the DNA helicase BACH1 and the CtBP-interacting protein (CtIP). The crystal structure of the BRCA1 BRCT repeats bound to the PTRVSpSPVFGAT phosphopeptide corresponding to residues 322-333 of human CtIP was determined at 2.5 A resolution. The peptide binds to a cleft formed by the interface of the two BRCTs in a two-pronged manner, with phospho-Ser327 and Phe330 anchoring the peptide through extensive contacts with BRCA1 residues. Several hydrogen bonds and salt bridges that stabilize the BRCA1-BACH1 complex are missing in the BRCA1-CtIP interaction, offering a structural basis for the approximately 5-fold lower affinity of BRCA1 for CtIP compared to that of BACH1, as determined by isothermal titration calorimetry. Importantly, the side chain of Arg1775 in the cancer-associated BRCA1 mutation M1775R sterically clashes with the phenyl ring of CtIP Phe330, disrupting the BRCA1-CtIP interaction. These results provide new insights into the molecular mechanisms underlying the dynamic selection of target proteins involved in DNA repair and cell cycle control by BRCA1 and reveal how certain cancer-associated mutations affect these interactions.     

PAR proteins direct asymmetry of the cell cycle regulators Polo-like kinase and Cdc25

Cell cycle lengths vary widely among different cells within an animal, yet mechanisms of cell cycle length regulation are poorly understood. In the Caenorhabditis elegans embryo, the first cell division produces two cells with different cell cycle lengths, which are dependent on the conserved partitioning-defective (PAR) polarity proteins. We show that two key cell cycle regulators, the Polo-like kinase PLK-1 and the cyclin-dependent kinase phosphatase CDC-25.1, are asymmetrically distributed in early embryos. PLK-1 shows anterior cytoplasmic enrichment and CDC-25.1 shows PLK-1-dependent enrichment in the anterior nucleus. Both proteins are required for normal mitotic progression. Furthermore, these asymmetries are controlled by PAR proteins and the muscle excess (MEX) proteins MEX-5/MEX-6, and the latter is linked to protein degradation. Our results support a model whereby the PAR and MEX-5/MEX-6 proteins asymmetrically control PLK-1 levels, which asymmetrically regulates CDC-25.1 to promote differences in cell cycle lengths. We suggest that control of Plk1 and Cdc25 may be relevant to regulation of cell cycle length in other developmental contexts.     

Arrest of cell cycle by Amida which is phosphorylated by Cdc2 kinase

Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Although previous studies have shown that Amida mRNA is predominantly expressed and developmentally regulated in rat testis and overexpression induces apoptosis, the function of Amida remains unclear. In this study, we found that overexpression of Amida inhibited cell growth. Flow cytometry analysis showed that Amida caused cell cycle inhibition in the S-phase and blocked cell cycle from entry into mitosis. Attempting to elucidate Amida effect on the cell cycle, we found that Amida was interacted with Cdc2 in mitosis and Amida's overexpression resulted in a decrease in Cdc2 kinase activity. In addition, Amida showed DNA-binding ability with DNA-affinity column chromatography. A region (aa, 76–189) between the two nuclear localization signals was found to be responsible for cell growth inhibition and DNA-binding activity, implying that DNA-binding activity may be necessary for Amida to repress cell cycle. Moreover, Amida was phosphorylated by Cdc2 kinase in vitro and Ser-180 of Amida was identified as the phosphorylation site. Furthermore, AmidaS180G (eliminate phosphorylation of Ser-180) showed stronger DNA-binding activity. Taken together, the data suggest that Amida may play an important role in cell cycle and may be partly regulated by Cdc2 kinase.     

Selective inhibition of bleomycin-induced G2 cell cycle checkpoint by simaomicin alpha

Human T-cell leukemia-derived Jurkat cells are known to be defective in the G1 checkpoint. DNA-damaging agent bleomycin arrests the cell cycle at G2 phase of Jurkat cells, and microtubule-acting colchicine arrests it at the M phase. Simaomicin alpha, an actinomycete metabolite, itself showed no effect on the cell cycle status of Jurkat cells at least up to 6.0 nM. However, the compound (0.6-6.0 nM) was found to abrogate the bleomycin-induced G2 arrest, yielding a drastic decrease in cells at the G2 phase and increase in cells at the subG1 and G1 phases. On the other hand, the compound did not show any effect on the colchicine-induced M phase arrest in Jurkat cells. Furthermore, the compound showed almost no effect on the cell cycle status of the bleomycin-treated or -untreated normal cell line HUVEC. These data suggested that simaomicin alpha disrupts the cell cycle G2 checkpoint of cancer cells selectively, leading to sensitization of cancer cells to anti-cancer reagents.     

Sibling rivalry in checkpoint control of cell cycle and DNA damage response

Comment on: Pabla N, et al. Proc Natl Acad Sci USA 2012; 109:197-202.     

Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery

A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.     

Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins

A search of the Dictyostelium genome project database (http://dictybase.org/db/cgi-bin/blast.pl) with nucleomorphin, a protein that regulates the nuclear number, predicted it to be encoded by a larger gene containing a putative breast cancer carboxy-terminus domain (BRCT). Using RT-PCR, Northern and Western blotting we have identified a differentially expressed, 2318 bp cDNA encoding a protein isoform of Dictyostelium NumA with an apparent molecular weight of 70 kDa that we have called NumB. It contains a single amino-terminal BRCT-domain spanning residues 125-201. Starvation of shaking cultures reduces NumA expression by approximately 88+/-5.6%, whereas NumB expression increases approximately 35+/-3.5% from vegetative levels. NumC, a third isoform that is also expressed during development but not growth, remains to be characterized. These findings suggest NumB may be a member of the BRCT-domain containing cell cycle checkpoint proteins.     

Cell cycle checkpoint proteins and cellular response to treatment by anticancer agents

The response of cancer cells to treatment with anticancer agents is mediated in part by proteins controlling both the cell cycle progression and the genomic integrity, including p53, p73 and checkpoint proteins chk1 and chk2. We here summarized the cellular functions of these proteins, their alterations in human tumors and the impact of their mutations/alterations on cellular response to treatment, Particular attention has been paid for those studies performed in isogenic cell systems, to minimize as much as possible interference by other alterations invariably present when different cell types are considered. Focus has also be given to the approaches taken to exploit the differential expression of these proteins between normal and tumor cells to improve the selectivity of treatment with anticancer agents.     

Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast

The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.     

The RSC chromatin-remodeling complex influences mitotic exit and adaptation to the spindle assembly checkpoint by controlling the Cdc14 phosphatase

Upon prolonged activation of the spindle assembly checkpoint, cells escape from mitosis through a mechanism called adaptation or mitotic slippage, which is thought to underlie the resistance of cancer cells to antimitotic drugs. We show that, in budding yeast, this mechanism depends on known essential and nonessential regulators of mitotic exit, such as the Cdc14 early anaphase release (FEAR) pathway for the release of the Cdc14 phosphatase from the nucleolus in early anaphase. Moreover, the RSC (remodel the structure of chromatin) chromatin-remodeling complex bound to its accessory subunit Rsc2 is involved in this process as a novel component of the FEAR pathway. We show that Rsc2 interacts physically with the polo kinase Cdc5 and is required for timely phosphorylation of the Cdc14 inhibitor Net1, which is important to free Cdc14 in the active form. Our data suggest that fine-tuning regulators of mitotic exit have important functions during mitotic progression in cells treated with microtubule poisons and might be promising targets for cancer treatment.