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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 Mitotic Inhibitor Mik1 Helps to Enforce the DNA Damage Checkpoint
Molecular biology of the cell, 2000Co-Authors: Beth A. Baber-furnari, Nicholas Rhind, Michael N. Boddy, Paul Shanahan, Antonia Lopez-girona, Paul RussellAbstract:The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Deltamik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple Mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.
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Nif1, a novel Mitotic Inhibitor in Schizosaccharomyces pombe
The EMBO journal, 1997Co-Authors: Paul RussellAbstract:In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.
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Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.
The EMBO Journal, 1993Co-Authors: Clare H Mcgowan, Paul RussellAbstract:Abstract In fission yeast, the M-phase inducing kinase, a complex of p34cdc2 and cyclin B, is maintained in an inhibited state during interphase due to the phosphorylation of Cdc2 at Tyr15. This phosphorylation is believed to be carried out primarily by the Wee1 kinase. In human cells the negative regulation of p34cdc2/cyclin B is more complex, in that Cdc2 is phosphorylated at two Inhibitory sites, Thr14 and Tyr15. The identities of the kinases that phosphorylate these sites are unknown. Since fission yeast Wee1 kinase behaves as a dual-specificity kinase in vitro, a popular hypothesis is that a human Wee1 homolog might phosphorylate p34cdc2 at both sites. We report here that a human gene, identified as a possible Wee1 homologue, blocks cell division when overexpressed in HeLa cells. This demonstrates functional conservation of the Wee1 Mitotic Inhibitor. Contrary to the dual-specificity kinase hypothesis, purified human Wee1 phosphorylates p34cdc2 exclusively on Tyr15 in vitro; no Thr14 phosphorylation was detected. Human and fission yeast Wee1 also specifically phosphorylate synthetic peptides at sites equivalent to Tyr15. Mutation of a critical lysine codon (Lys114) believed to be essential for kinase activity abolished both the in vivo Mitotic Inhibitor function and in vitro kinase activities of human Wee1. These results conclusively prove that Wee1 kinases inhibit mitosis by directly phosphorylating p34cdc2 on Tyr15, and strongly indicate that human cells have independent kinase pathways directing the two Inhibitor phosphorylations of p34cdc2.
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dual functions of cdc6 a yeast protein required for dna replication also inhibits nuclear division
The EMBO Journal, 1992Co-Authors: Avelino Bueno, Paul RussellAbstract:Abstract The Saccharomyces cerevisiae gene CDC6, whose protein product is required for DNA replication, is transcribed only in late G1 and S phases. We have discovered a critical reason why CDC6 expression is regulated in this fashion. Constitutive CDC6 transcription greatly delayed the initiation of M phase without effecting the G1-S transition or growth rate. This occurred in both fission and budding yeasts. The CDC6-induced M phase delay was dependent on the wee1/mik1 Mitotic Inhibitor kinases and was greatly accentuated in strains defective for the cdc25/MIH1 Mitotic inducer phosphatases, indicating that CDC6 indirectly inhibits activation of the p34cdc2/CDC28 M phase kinase. Thus CDC6 appears to have an important and perhaps unique dual role in S phase, it is first required for the initiation of DNA replication and then actively participates in the suppression of nuclear division.
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Fission yeast p107wee1 Mitotic Inhibitor is a tyrosine/serine kinase.
Nature, 1991Co-Authors: Carol Featherstone, Paul RussellAbstract:The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.
Carol Featherstone - One of the best experts on this subject based on the ideXlab platform.
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Fission yeast p107wee1 Mitotic Inhibitor is a tyrosine/serine kinase.
Nature, 1991Co-Authors: Carol Featherstone, Paul RussellAbstract:The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.
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fission yeast p107wee1 Mitotic Inhibitor is a tyrosine serine kinase
Nature, 1991Co-Authors: Carol Featherstone, Paul RussellAbstract:The fission yeast wee1+ gene product is a dose-dependent, negative regulator of entry into mitosis. wee1+ encodes a protein of relative molecular mass 107,000 (Mr 107K), the C-terminal third of which has strong similarities with the serine/threonine protein kinase family. Here we report that p107wee1 immune complexes phosphorylate p107wee1 equally on serine and tyrosine residues, and also phosphorylate an exogenous substrate, angiotensin II, on tyrosine. Both kinase activities are attributable to p107wee1 because they are also observed when wee1+ is expressed in heterologous systems; both are abolished by a point mutation in the ATP-binding domain, and both behave like an asymmetric monomer of Mr114K on gel filtration and density-gradient centrifugation. Thus the wee1+ gene product is representative of a novel class of protein kinase that phosphorylates both serine and tyrosine residues.
Dina Moustafa - One of the best experts on this subject based on the ideXlab platform.
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modulation of early Mitotic Inhibitor 1 emi1 depletion on the sensitivity of parp Inhibitors in brca1 mutated triple negative breast cancer cells
PLOS ONE, 2021Co-Authors: Dina Moustafa, Maha Abd R Elwahed, Hanaa H Elsaid, Jeffrey D ParvinAbstract:Triple negative breast cancer (TNBC) represents approximately 10-15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase Inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 Inhibitor. Notably, increasing the RAD51 protein expression only recapitulated some, but not all, of the effects of EMI1 depletion in conferring to the cell resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking Mitotic entry. We found that combining CHK1 Inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.
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modulation of early Mitotic Inhibitor 1 emi1 depletion on the sensitivity of parp Inhibitors in brca1 mutated triple negative breast cancer cells
bioRxiv, 2020Co-Authors: Jeffrey D Parvin, Dina Moustafa, Maha Abd R Elwahed, Hanaa H ElsaidAbstract:Triple negative breast cancer (TNBC) represents approximately 10–15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase Inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 Inhibitor. Surprisingly, EMI1 depletion also led to resistance to a MEK Inhibitor, though this Inhibitor blocks cells in G1 phase of the cell cycle and would not be expected to be sensitive to EMI1 levels. Notably, increasing the RAD51 protein expression only partially recapitulated the effects of EMI1 depletion in causing resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking Mitotic entry. We found that combining CHK1 Inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.
Peter K Jackson - One of the best experts on this subject based on the ideXlab platform.
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prophase i arrest and progression to metaphase i in mouse oocytes are controlled by emi1 dependent regulation of apc cdh1
Journal of Cell Biology, 2007Co-Authors: Petros Marangos, Peter K Jackson, Emmy W Verschuren, Ruby Chen, John CarrollAbstract:Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early Mitotic Inhibitor 1 (Emi1), an Inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1–Cul1–F-box/βTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1–GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1–GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APCCdh1.
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a role for the anaphase promoting complex Inhibitor emi2 xerp1 a homolog of early Mitotic Inhibitor 1 in cytostatic factor arrest of xenopus eggs
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Jeffrey J Tung, David V Hansen, Alexander V Loktev, Matthew K Summers, John R Adler, Peter K JacksonAbstract:Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC Inhibitor early Mitotic Inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCFβTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC Inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.
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A role for the anaphase-promoting complex Inhibitor Emi2/XErp1, a homolog of early Mitotic Inhibitor 1, in cytostatic factor arrest of Xenopus eggs
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Jeffrey J Tung, David V Hansen, Alexander V Loktev, Matthew K Summers, John R Adler, Kenneth H K Ban, Peter K JacksonAbstract:Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC Inhibitor early Mitotic Inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCFβTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC Inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.
Helen Piwnica-worms - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation and inactivation of the Mitotic Inhibitor Wee1 by the nim1/cdr1 kinase
Nature, 1993Co-Authors: Laura L Parker, Sarah A Walter, Paul G. Young, Helen Piwnica-wormsAbstract:THE G2-M phase transition in eukaryotes is regulated by the synergistic and opposing activities of a cascade of distinct protein kinases and phosphatases. This cascade converges on Cdc2, a serine/threonine protein kinase required for entry into mitosis (reviewed in ref. 1). In the fission yeast Schizosaccharomyces pombe, inactivation of the Cdc2/cyclin B complex is achieved by phosphorylation of tyrosine 15 by Weel (refs 2, 3). The action of the Weel kinase is opposed by the action of the Cdc25 phosphatase, which dephosphorylates Cdc2 on tyrosine 15, thereby activating the Cdc2/cyclin B complex4–9. Much less is known about the regulatory signals upstream of cdc25 and wee1. Genetics indicate that the Mitotic inducer nim1/cdr1 acts upstream of wee1, possibly as a negative regulator of wee1 (refs 10, 11). To characterize the nim1/cdr1 protein (Nim1), we have overproduced it in both bacterial and baculoviral expression systems. We report that Nim1 possesses intrinsic serine-kinase, threonine-kinase and tyrosine-kinase activities. Co-expression of the Nim1 and Wee1 kinases in insect cells results in the phosphorylation of Weel and therefore a shift in its electrophoretic mobility on SDS–polyacrylamide gels. When Weel is phosphorylated, its ability to phosphorylate Cdc2 on tyrosine 15 is inhibited; treatment with phosphatase restores this kinase activity. Furthermore, purified bacterially produced Nim1 kinase directly phosphorylates and inactivates Weel in vitro. These results show that nim1/cdr1 functions as a positive regulator of mitosis by directly phosphorylating and inactivating the Mitotic Inhibitor Weel.
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p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: L. L. Parker, Sue Atherton-fessler, Helen Piwnica-wormsAbstract:p107wee1 is a protein kinase that functions as a dose-dependent Inhibitor of mitosis through its interactions with p34cdc2 in Schizosaccharomyces pombe. To characterize the kinase activity of p107wee1, its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37wee1KD) was determined to be approximately 37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37wee1KD, demonstrating that p107wee1 is a dual-specificity kinase. In vitro, p107wee1 phosphorylated p34cdc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34cdc2 nor a peptide containing Tyr-15 was able to substitute for the p34cdc2/cyclin complex in this assay. Furthermore, the phosphorylation of p34cdc2 by p107wee1 in vitro inhibited the histone H1 kinase activity of p34cdc2. These results indicate that p107wee1 functions as a Mitotic Inhibitor by directly phosphorylating p34cdc2 on Tyr-15 and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.