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Query: UNIPROT:P06889 (Mol)
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During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint.
Mol Cell 2006 Aug 04
PMID:SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response. 1688 22

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.
Mol Biol Cell 2006 Oct
PMID:Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins. 1688 16

Outer dense fiber 2 (Odf2) was initially identified as a major component of sperm tail cytoskeleton and later was suggested to be a widespread component of centrosomal scaffold that preferentially associates with the appendages of the mother centrioles in somatic cells. Here we report the identification of two Odf2-related centrosomal components, hCenexin1 and hCenexin1 variant 1, that possess a unique C-terminal extension. Our results showed that hCenexin1 is the major isoform expressed in HeLa cells, whereas hOdf2 is not detectably expressed. Mammalian polo-like kinase 1 (Plk1) is critical for proper mitotic progression, and its association with the centrosome is important for microtubule nucleation and function. Interestingly, depletion of hCenexin1 by RNA interference (RNAi) delocalized Plk1 from the centrosomes and the C-terminal extension of hCenexin1 was crucial to recruit Plk1 to the centrosomes through a direct interaction with the polo-box domain of Plk1. Consistent with these findings, the hCenexin1 RNAi cells exhibited weakened gamma-tubulin localization and chromosome segregation defects. We propose that hCenexin1 is a critical centrosomal component whose C-terminal extension is required for proper recruitment of Plk1 and other components crucial for normal mitosis. Our results further suggest that the anti-Odf2 immunoreactive centrosomal antigen previously detected in non-germ line cells is likely hCenexin1.
Mol Cell Biol 2006 Nov
PMID:Requirement of hCenexin for proper mitotic functions of polo-like kinase 1 at the centrosomes. 1696 75

The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.
Mol Cell 2006 Nov 03
PMID:Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. 1718 28

Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the Nterminus region, whereas the non-catalytic region in the Cterminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domainspecific target proteins of Plk1, we constructed an Nterminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and Ctermini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.
J Biochem Mol Biol 2006 Nov 30
PMID:Screening of domain-specific target proteins of polo-like kinase 1: construction and application of centrosome/kinetochore-specific targeting peptide. 1712 6

A new centromeric protein termed PBIP1 was identified that recruits Plk1 to the kinetochores. In the November 3 issue of Molecular Cell, show that Plk1 phosphorylates PBIP1 on threonine 78, creating its own high-affinity docking site for the polo-box domain (PBD).
Mol Cell 2006 Nov 17
PMID:Grabbing Plk1 by the PBD. 1708 91

Polo-like kinase 1(Plk1) has been reported to be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during mammalian early embryonic mitosis. In the present study, we examined the expression of Plk1 at protein and mRNA level in mouse fertilized eggs by Western blot and RT-PCR. We also examined the kinase activity of Plk1. At various developmental phases of mouse one-cell stage embryos, both the protein and the mRNA of Plk1 were uniformly distributed; but the kinase activity of Plk1 increased at G2/M phase and decreased at the end of M phase. At the meantime, the phosphorylation of Tyr 15 of Cdc2 was inhibited at M phase. To investigate its function in mammalian fertilized eggs further, we used specific short hairpin RNAs (shRNA) and scytonemin, the putative inhibitor of Plk1 to suppress the activity of Plk1 in mouse fertilized eggs. Upon blockage of the activation of with Plk1 shRNA and scytonemin in mouse one-cell stage embryos, the cleavage rate decreased and the phosphorylation level of Tyr 15 of Cdc2 increased. These results imply that the Plk1 may regulate cell cycle progression of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2.
Mol Reprod Dev 2007 Oct
PMID:Polo-like kinase 1 may regulate G2/M transition of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2. 1734 25

Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.
Mol Cell Biol 2007 Jul
PMID:DNA damage checkpoints inhibit mitotic exit by two different mechanisms. 1748 42

Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.
J Mol Biol 2007 Sep 14
PMID:Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling. 1766 93

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.
Mol Biol Cell 2007 Oct
PMID:Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast. 1769 98


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