Kumagai A, Guo Z, Emami KH, Wang SX, Dunphy WG.

GM-43974 NIGMS NIH HHS , R01 GM043974 NIGMS NIH HHS , R37 GM043974 NIGMS NIH HHS

	Figure 2. Xchk1 phosphorylates Cdc25 on Ser-287 and mediates binding of 14-3-3. (A) Recombinant His6–Xchk1 (lane 1) and His6–Xchk1-N135A (lane 2) were purified from baculovirus-infected insect cells, subjected to SDS-PAGE, and then stained with Coomassie blue. (B) Xchk1 phosphorylates Cdc25 efficiently in vitro. His6–Xchk1 (lanes 1 and 3) and His6–Xchk1-N135A (lanes 2 and 4) were incubated with either His6–Cdc25-WT (lanes 1 and 2) or His6–Cdc25-S287A (lanes 3 and 4) in kinase buffer containing 10 μM [γ-32P]ATP. Reactions were terminated with gel sample buffer and subjected to SDS-PAGE and autoradiography. (C) Xchk1 phosphorylates Cdc25 at Ser-287 and other sites. His6–Cdc25-WT and His6–Cdc25-S287A that had been incubated with His6–Xchk1 in the presence of 10 μM [γ-32P]ATP were processed for tryptic phosphopeptide mapping. Arrows, position of the peptide containing Ser-287; dots, origins in the lower left corner of each map. (D) Specific phosphorylation by His6– Xchk1 of a fragment of Cdc25 containing the 14-3-3 binding site. GST–Cdc25(254–316)-WT (lane 1) or GST–Cdc25(254–316)- S287A (lane 2) were incubated with His6–Xchk1 in the presence of [γ-32P]ATP and subjected to SDS-PAGE and autoradiography. Arrow, position of the wild-type and mutant GST fusion proteins. (E) Phosphorylation of Cdc25 by Xchk1 creates a 14-3-3 binding site. Bacterially-expressed GST–Cdc25-WT (lanes 1–3) and GST–Cdc25-S287A (lane 4) bound to glutathione agarose were incubated with His6–Xchk1 (lanes 2 and 4), His6–Xchk1-N135A (lane 3), or neither protein (lane 1) in kinase buffer containing 1 mM ATP at 23°C for 30 min. His6–14-3-3ε was then added to the mixture, and the incubation was continued at 4°C for an additional 30 min. At the end of the incubation, proteins specifically bound to the beads were eluted with glutathione and subjected to SDS-PAGE and immunoblotting with either anti-Cdc25 antibodies (top) or anti–14-3-3ε antibodies (bottom).
	Figure 3. Immunodepletion of Xchk1 compromises checkpoint control in Xenopus egg extracts. (A) Immunodepletion of Xchk1 from Xenopus egg extracts. An M phase extract (lane 1) was treated with anti-Xchk1 antibodies (lanes 3 and 4) or control antibodies (lane 2) bound to protein A beads as described in Materials and Methods. In lane 4, His6–Xchk1 protein was added back to the Xchk1-depleted extract to a final concentration of 2 ng/μl. Samples (lanes 1–4 ) were processed for immunoblotting with anti-Xchk1 antibodies. (B) Effect of immunodepletion of Xchk1 on egg extracts lacking unreplicated DNA. M phase extracts were treated with anti-Xchk1 antibodies (•, ▪) or control antibodies (▴). In one set of samples (▪), His6–Xchk1 was added back to a concentration of 2 ng/μl. The extracts were activated with calcium, and the timing of NEB (nuclear envelope breakdown) was monitored by microscopy. (C) Effect of immunodepletion of Xchk1 on egg extracts containing unreplicated DNA. Extracts that had been treated with either anti-Xchk1 antibodies (•, ○, ▪) or control antibodies (▴, ▵) were incubated with 1,000 sperm nuclei/μl and 100 μg/ml aphidicolin in the presence (○, ▵) or absence (•, ▴, ▪) of 5 mM caffeine. In one case (▪), 2 ng/μl His6– Xchk1 was added back to the Xchk1-depleted extract.
	Figure 4. Cdc25 undergoes phosphorylation of Ser-287 and binds to 14-3-3ε in Xchk1-depleted extracts. (A) The 14-3-3ε protein binds to Cdc25 in Xchk1-depleted extracts. The 14-3-3ε protein was immunoprecipitated from a mock-depleted extract (lane 1), an Xchk1-depleted extract (lane 2), or an Xchk1-depleted extract supplemented with 2 ng/μl His6–Xchk1 protein (lane 3). For lanes 4–7, an M phase extract (lane 4) or interphase extracts (lanes 5–7) containing 1,000 sperm nuclei/μl (lane 5),  3,000 sperm nuclei/μl and 100 μg/ml aphidicolin (lane 6), or 3,000 UV-damaged sperm nuclei per μl (lane 7) were immunoprecipitated with anti–14-3-3ε antibodies. All extracts contained 100 μg/ml cycloheximide. In all cases, the anti–14-3-3ε immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-Cdc25 (top) or anti–14-3-3ε (bottom) antibodies. APH, aphidicolin. (B) Mock-depleted extracts (lanes 1 and 2) and Xchk1-depleted extracts (lane 3) containing 1,000 sperm nuclei/μl were assayed for Ser-287–specific kinase activity using either GST–Cdc25(254– 316)-WT (lanes 2 and 3) or GST–Cdc25(254–316)-S287A (lane 1) as the substrate.
	Figure 5. Exogenously added His6–Xchk1 delays mitosis in egg extracts in a caffeine-resistant manner. (A) His6–Xchk1 (•, ▴), His6–Xchk1-N135A (▪), or buffer only (○) was added to egg extracts containing 1,000 sperm nuclei/μl in the presence (▴) or absence (•, ▪, ○) of 5 mM caffeine. The timing of NEB was monitored by microscopy. Recombinant Xchk1 proteins were added to a final concentration of 10 ng/μl. Typically, caffeine did not affect mitotic timing significantly in the extracts containing buffer only or His6–Xchk1-N135A.
	Figure 6. Xchk1 is modified when the DNA replication and DNA damage checkpoints are activated. (A) 35S-labeled Xchk1 was incubated in interphase egg extracts containing 3,000 sperm nuclei/μl (lane 1), 3,000 UV-damaged sperm nuclei/μl (lanes 2 and 3), or 3,000 sperm nuclei/μl and 100 μg/ml aphidicolin (lanes 4 and 5) in the presence (lanes 3 and 5) or absence (lanes 1, 2, and 4) of 5 mM caffeine. After 100 min of incubation at 23°C, 2 μl of each extract was taken for SDS-PAGE and autoradiography (top). Alternatively, 50 μl of each extract was centrifuged through a sucrose solution to isolate the nuclear fraction (bottom), which was also subjected to SDS-PAGE and autoradiography. APH, aphidicolin. (B) 35S-labeled Xchk1 was immunoprecipitated from interphase extracts containing 3,000 sperm nuclei/ μl and 100 μg/ml aphidicolin. The immunoprecipitates were incubated in phosphatase buffer containing no addition (lane 1), protein phosphatase 2A (lane 2), or protein phosphatase 2A and 3 μM okadaic acid (lane 3). PP2A, protein phosphatase 2A; OA, okadaic acid. (C) 35S-labeled Xchk1 (lanes 1–4) and Xchk1– N135A (lanes 5–8) were incubated for 100 min in either control-depleted (lanes 1, 2, 5, and 6) or Xchk1-depleted (lanes 3, 4, 7, and 8) extracts in the presence of either 3,000 sperm nuclei/μl (lanes 1, 3, 5, and 7) or 3,000 UV-damaged sperm nuclei/μl (lanes 2, 4, 6, and 8). Nuclear fractions were isolated as described above and subjected to SDS-PAGE and autoradiography.
	Figure 7. Model for checkpoint regulation in Xenopus egg extracts. Refer to text for details.

al-Khodairy, Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. 1994, Pubmed

al-Khodairy, Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast. 1994, Pubmed
Bentley, The Schizosaccharomyces pombe rad3 checkpoint gene. 1996, Pubmed
Boddy, Replication checkpoint enforced by kinases Cds1 and Chk1. 1998, Pubmed
Carr, Checkpoints take the next step. 1996, Pubmed
Carr, Control of cell cycle arrest by the Mec1sc/Rad3sp DNA structure checkpoint pathway. 1997, Pubmed
Carr, The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'. 1995, Pubmed
Cliby, Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. 1998, Pubmed
Dasso, Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. 1990, Pubmed , Xenbase
Dunphy, The cdc25 protein contains an intrinsic phosphatase activity. 1991, Pubmed
Elledge, Cell cycle checkpoints: preventing an identity crisis. 1996, Pubmed
Featherstone, Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase. 1991, Pubmed
Flaggs, Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes. 1997, Pubmed
Fogarty, The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity. 1997, Pubmed
Francesconi, p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast. 1997, Pubmed
Furnari, Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase. 1997, Pubmed
Gautier, cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. 1991, Pubmed , Xenbase
Hartwell, Checkpoints: controls that ensure the order of cell cycle events. 1989, Pubmed
Hoffmann, Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis. 1993, Pubmed , Xenbase
Igarashi, Wee1(+)-like gene in human cells. 1991, Pubmed
Izumi, Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity. 1995, Pubmed , Xenbase
Izumi, Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. 1992, Pubmed , Xenbase
Keegan, The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes. 1996, Pubmed
King, Mitosis in transition. 1994, Pubmed
Kumagai, Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. 1996, Pubmed , Xenbase
Kumagai, 14-3-3 proteins act as negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts. 1998, Pubmed , Xenbase
Kumagai, Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors. 1995, Pubmed , Xenbase
Kumagai, Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. 1992, Pubmed , Xenbase
Morgan, Cyclin-dependent kinases: engines, clocks, and microprocessors. 1997, Pubmed
Mueller, Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15. 1995, Pubmed , Xenbase
Mueller, Cell cycle regulation of a Xenopus Wee1-like kinase. 1995, Pubmed , Xenbase
Murray, Cell cycle extracts. 1991, Pubmed
Murray, The genetics of cell cycle checkpoints. 1995, Pubmed
Nurse, Checkpoint pathways come of age. 1997, Pubmed
O'Connell, Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. 1997, Pubmed
Parker, p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. 1992, Pubmed
Paulovich, A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. 1995, Pubmed
Peng, Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. 1997, Pubmed
Peng, C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. 1998, Pubmed
Poon, The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells. 1997, Pubmed
Rhind, Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. 1997, Pubmed
Saka, Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. 1997, Pubmed
Sanchez, Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. 1996, Pubmed
Sanchez, Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. 1997, Pubmed
Schlegel, Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. 1986, Pubmed
Sibon, DNA-replication checkpoint control at the Drosophila midblastula transition. 1997, Pubmed
Smith, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. 1988, Pubmed
Strausfeld, Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. 1991, Pubmed
Sun, Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. 1996, Pubmed
Walworth, rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. 1996, Pubmed
Walworth, Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. 1993, Pubmed
Watanabe, Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle. 1995, Pubmed
Weinert, A DNA damage checkpoint meets the cell cycle engine. 1997, Pubmed
Westphal, Cell-cycle signaling: Atm displays its many talents. 1997, Pubmed
Yaffe, The structural basis for 14-3-3:phosphopeptide binding specificity. 1997, Pubmed