Conservation of the Chk1 Checkpoint Pathway in Mammals: Linkage of DNA Damage to Cdk Regulation Through Cdc25

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We made recombinant baculovirus encoding glutathione-S-transferase (GST) fusion proteins to hChk1 (GST-hChk1) or to a mutation of hChk1 in which Asp at position 130 was mutated to Ala [GST-hChk1(D130A)] (pYS71). These recombinants were made by introducing an Nde I site at the first ATG codon of the hChk1 open reading frame using PCR, and subcloning the hChk1 cDNA as an Nde I–Eco RI fragment into pGEX2Tcs, generating pYS45. The Xba I–Eco RI fragment from pYS45 containing GST-hChk1 was subcloned into PVL1393, which was cut with Xba I and Eco RI, generating pYS63. The GST-hChk1(D130A) mutant was generated by PCR, and the Xho I–Xmn I fragment containing the mutation was used to replace the wild-type fragment, generating pYS64. The GST-hChk1(D130A) fragment from pYS64 was sublconed into the baculovirus transfer vector by using the Univector plasmid fusion strategy (X. Liu and S. Elledge, unpublished results). Viruses were generated by standard methods (Baculogold, Pharmingen). Recombinant GST-hChk1 protein was isolated from infected Hi5 insect cells on glutathione (GSH) agarose. Cdc25C was cloned into pET15b (Novagen) and purified as outlined by the manufacturer.

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Kinase reactions contained GST-hChk1 bound to GSH agarose and either His₆-hCdc25C GST-hCdc25A, GST-hCdc25B, GST-hCdc25C, or GST-hCdc25C- (200–256) (amino acids 200 to 256 of Cdc25). Kinase reactions contained 1 to 3 μg of GST-hChk1 or GST-hChk1(D130A) protein on beads and soluble substrate in 20 mM Hepes (pH 7.4), 10 mM MgCl₂, 10 mM MnCl₂, 2 μM adenosine triphosphate (ATP), and 15 μCi of [γ-³²P]ATP for 30 min at 30°C. To determine the site on Cdc25C phosphorylated by hChk1, we carried out kinase reactions in a buffer consisting of 50 mM tris (pH 7.4), 10 mM MgCl₂, 10 μM ATP, 1 mM dithiothreitol (DTT), and 10 μCi of [γ-³²P]ATP. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and visualized by autoradiography. The nitrocellulose membrane containing His₆-Cdc25C was excised, blocked with 0.5% polyvinylpyrrolidone (PVP-40) in 100 mM acetic acid for 30 min at 37^oC, washed six times with water, and digested with TPCK trypsin (Worthington) at a final concentration of 30 mg/ml in 0.1 M NH₄CO₃ (pH 8.0). Further digestion on selected high-pressure liquid chromatography (HPLC) fractions was performed with 2 units of proline-specific endopeptidase (ICN) in 0.1 M sodium phosphate, 5 mM EDTA (pH 7.4) at 37^oC for 16 hours. Samples were acidified in 1% trifluoroacetic acid (TFA) and loaded onto a Vydac C18 column (25 cm by 0.46 cm inner diameter). Reverse-phase HPLC was performed at 37^oC. Reactions were loaded in 0.1% TFA (buffer A) and eluted with a gradient from 0 to 60% buffer B (90% acetonitrile, 0.095% TFA). Fractions were collected at 0.5-min intervals up to 90 min and counted for radioactivity. Selected fractions were immobilized on Sequenlon-AA membrane discs (Millipore) for NH₂-terminal sequencing. Manual Edman degradation was done as described (21) with a coupling and cleavage temperature of 55^oC.

Y. Sanchez et al., data not shown.

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We thank A. Baldini for assistance with mapping; T. Carr for sharing unpublished information; P. Sen for making the S216A mutants of Cdc25C; N. Walworth, W. Harper, M. Huang, and J. Bachant for helpful comments; and D. Leibham and J. Thompson for technical assistance. Support was by a NIH postdoctoral fellowship GM17763 to Y.S. and a NIH grant GM44664 to S.J.E. H.P.-W. is an Associate Investigator of the Howard Hughes Medical Institute. S.J.E. is a Pew Scholar in the Biomedical Sciences and an Investigator of the Howard Hughes Medical Institute.