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.

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