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Paul Russell - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Schizosaccharomyces pombe Wee1 Tyrosine Kinase
Journal of Biological Chemistry, 1997Co-Authors: Rosa Aligue, Lin Wu, Paul RussellAbstract:Abstract Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of ∼350 amino acids preceded by a N-terminal domain of ∼550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363–408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.
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SpWee1: Wee1 protein kinase (S. pombe)
The Protein Kinase FactsBook, 1995Co-Authors: Paul RussellAbstract:Wee1 functions to delay the onset of mitosis until cells have grown to a sufficient size. On the basis of sequence comparisons, Wee1 was predicted to be a protein-serine/threonine kinase, but it inhibits mitosis by phosphorylating the SpCdc2 protein kinase on Tyr15. A human homologue (Wee1) and Saccharomycer cerevisiae (Swe1) homologues have been described. The enzyme is most conveniently assayed by transfer of radioactivity from [γ32P]ATP to angiotensin II or Cdc2. Wee1 can be inactivated by phosphorylation carried out by Nim1.
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cell cycle regulation of human Wee1
The EMBO Journal, 1995Co-Authors: Clare H Mcgowan, Paul RussellAbstract:Abstract Wee1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human Wee1. Endogenous Wee1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human Wee1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that Wee1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. Wee1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating Wee1. Wee1 activity is strongly suppressed during M phase, suggesting that negative regulation of Wee1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase Wee1 is re-activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that Wee1 is inhibited by a mechanism that requires protein phosphorylation.
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nim1 kinase promotes mitosis by inactivating Wee1 tyrosine kinase
Nature, 1993Co-Authors: Lin Wu, Paul RussellAbstract:IN most species, including the fission yeast Schizosaccharomyces pombe, the Cdc2/cyclin B mitosis-inducing kinase is maintained in an inhibited state during interphase as a result of phosphorylation of a tyrosine residue in the ATP-binding region of Cdc2 (refs 1–3). This site is phosphorylated by Wee1 kinase4–9 and dephosphorylated by Cdc25 phosphatase10–15. In fission yeast an additional element of the G2/M control Nim1/Cdr1 kinase, has been identified which functions as a potent mitotic inducer16,17. These studies suggested that Nim1 acts by inhibiting Wee1, perhaps by direct phosphorylation. Consistent with this model, we report here that Wee1 is hyperphosphorylated in cells that overproduce Nim1. Likewise, Wee1 phosphorylation is reduced in nim1− cells. Highly purified Nim1 kinase phosphorylates Wee1 in vitro, resulting in strong inhibition of Wee1 kinase. These observations show that Nim1 promotes the onset of mitosis by inhibiting Wee1.
William G Dunphy - One of the best experts on this subject based on the ideXlab platform.
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positive regulation of Wee1 by chk1 and 14 3 3 proteins
Molecular Biology of the Cell, 2001Co-Authors: Akiko Kumagai, William G DunphyAbstract:Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (XWee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that XWee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to XWee1 occurs during interphase, but not M-phase, and requires phosphorylation of XWee1 on Ser-549. A mutant of XWee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type XWee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of XWee1. In cell-free kinase assays, Xchk1 phosphorylates XWee1 on Ser-549. The results of experiments in which XWee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous XWee1 with recombinant XWee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of XWee1.
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cell cycle regulation of a xenopus Wee1 like kinase
Molecular Biology of the Cell, 1995Co-Authors: Paul R Mueller, Thomas R. Coleman, William G DunphyAbstract:Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr- 15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.
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Two distinct mechanisms for negative regulation of the Wee1 protein kinase.
The EMBO Journal, 1993Co-Authors: Zhaohua Tang, Thomas R. Coleman, William G DunphyAbstract:Abstract The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.
Hiroto Okayama - One of the best experts on this subject based on the ideXlab platform.
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- Wee1: Wee1 protein kinase (vertebrates)
The Protein Kinase FactsBook, 1995Co-Authors: Hiroto Okayama, Makato Igarashi, Akihisa NagataAbstract:In eukaryotes, mitosis is initiated by the activation of the cyclin-associated Cdc2 kinase. In the S and G2 phases of the cell cycle, this kinase is inactivated by phosphorylation at Tyr15. Wee1 kinase catalyzes this phosphrylation. At the end of G2 phase, Cdc2 undergoes dephosphorylation and concomitant activation, which is catalyzed by specific phosphotase(s), Cdc25. Wee1 resembles SpWee1 and complements the Schizosaccharomyce pombe Wee1 mutant. Structurally, this kinase belongs to the serine/threonine PK family, yet possesses tyrosine kinase activity. Human and rat Wee1 kinase cDNAs have been cloned and sequenced. The only known physiological substrate is Cdc2 associated with cyclin B. The enzyme is assayed by transfer of radioactivity from [γ32P]ATP to cyclin B-associated Cdc2. The Wee1 gene is expressed in a cell cycle-dependent manner: the transcript begins to appear in S phase, peaks at G2 phase, and decreases thereafter. The enzyme is localized in the nucleus.
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Wee1 like gene in human cells
Nature, 1991Co-Authors: Makoto Igarashi, Akihisa Nagata, S Jinno, Kimihiko Suto, Hiroto OkayamaAbstract:THE Wee1+ gene is a mitotic inhibitor controlling the G2 to M transition of the fission yeast Schizosaccharomyces pombe1 and encodes a protein kinase with both serine- and tyrosine-phosphorylating activities2. We have cloned a human gene (WEElHu) similar to weel+ by transcomplementation of a yeast mutant3,4. WEElHu encodes a protein homologous to the S. pombe weel+ and mik1+ (a functionally redundant sibling of weel+)5 kinases and effectively rescues a Wee1 mutation. We report here that overexpression of Wee1Hu in fission yeast generates very elongated cells as a result of inhibition of the G2-M transition in the cell cycle. In addition, we detected a 3-kilobase-long Wee1Hu messenger RNA in all the human cell lines we examined. We conclude that a Wee1+-like gene exists and is expressed in human cells.
Hiroyuki Osada - One of the best experts on this subject based on the ideXlab platform.
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cyclin dependent kinase cdk phosphorylation destabilizes somatic Wee1 via multiple pathways
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Nobumoto Watanabe, Harumi Arai, Tony Hunter, Junichi Iwasaki, Masaaki Shiina, Kazuhiro Ogata, Hiroyuki OsadaAbstract:At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCFβ-TrCP. The F-box protein β-TrCP (β-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to β-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved β-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in β-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to β-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of β-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second β-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.
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m phase kinases induce phospho dependent ubiquitination of somatic Wee1 by scfβ trcp
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Nobumoto Watanabe, Harumi Arai, Yoshifumi Nishihara, Makoto Taniguchi, Naoko Watanabe, Tony Hunter, Hiroyuki OsadaAbstract:Abstract Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is down-regulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the β-transducin repeat-containing protein 1/2 (β-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCFβ-TrCP1/2) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for β-TrCP, recognition of Wee1A by β-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for β-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by β-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCFβ-TrCP1/2 in vitro. Mutation of these residues or depletion of β-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that β-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide.
Khanh Do - One of the best experts on this subject based on the ideXlab platform.
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phase i study of single agent azd1775 mk 1775 a Wee1 kinase inhibitor in patients with refractory solid tumors
Journal of Clinical Oncology, 2015Co-Authors: Khanh Do, Deborah Wilsker, Jiuping Ji, Jennifer Zlott, Tomoko Freshwater, Robert J Kinders, Jerry E Collins, Alice P Chen, J H Doroshow, S KummarAbstract:Purpose Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AX (γH2AX) levels in paired tumor biopsies. Patients and Methods AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. Results Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Co...
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Wee1 kinase as a target for cancer therapy
Cell Cycle, 2013Co-Authors: Khanh Do, James H Doroshow, Shivaani KummarAbstract:Wee1, a protein kinase, regulates the G2 checkpoint in response to DNA damage. Preclinical studies have elucidated the role of Wee1 in DNA damage repair and the stabilization of replication forks, supporting the validity of Wee1 inhibition as a viable therapeutic target in cancer. MK-1775, a selective and potent small-molecule inhibitor of Wee1, is under clinical development as a potentiator of DNA damage caused by cytotoxic chemotherapies. We present a review of the role of Wee1 in the cell cycle and DNA replication and summarize the clinical development to date of this novel class of anticancer agents.
