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doi: 10.1242/10.1242/jcs.00133

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Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest

Holly Capasso1,2, Carmela Palermo1,2, Shanhong Wan1,2, Hui Rao1, Ulrik P. John4, Matthew J. O'Connell4,5 and Nancy C. Walworth1,2,3,*

1 Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
2 Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Graduate School of Biomedical Sciences and Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
3 Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
4 Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, St Andrews Place, East Melbourne 3002, Australia
5 Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia



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  		Fig. 1. Mutating serine 345 to alanine abolishes the change in Chk1 mobility in response to CPT and UV light and confers UV sensitivity. (A) CLUSTAL W alignment of the non-catalytic domains of human, fission yeast, frog and mouse Chk1. Numbers refer to amino acids in the fission yeast protein. (B) NW223 (Chk1), NW457 (T323A), NW444 (S345A) and NW481 (S367A) cells grown to mid-log phase were treated with CPT, lysed, subjected to SDS-PAGE and immunoblotted with 12CA5 anti-HA antibody. (C) Strains shown in B were grown to mid-log phase, spread on agar plates, treated with 100 J/m2 UV light, lysed, subjected to SDS-PAGE, and immunoblotted with 12CA5 anti-HA antibody. (D) NW223 (chk1+), NW158 (chk1::ura4), NW457 (chk1T323A), NW444 (chk1S34A), and NW481 (chk1S36A) cells were grown to mid-log phase, resuspended to 1x107 cells/ml, spotted onto agar plates, and incubated at 30°C for approximately 3 days.

 


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  		Fig. 2. Failure to undergo the DNA damage-induced mobility shift correlates with lack of nuclear localization. (A) Strains expressing the indicated catalytic domain mutants with three HA tags at the C-terminus were expressed from the chk1 promoter on the plasmid pSP1 in a chk1::ura4 disruption strain (NW158). Cells in mid-log phase were exposed to 20 µM CPT for 2 hours. Lysates were prepared, separated by SDS-PAGE and examined by western blot analysis for the HA tag on Chk1. Data is not shown for Chk1G108S, but it shows no evidence of a mobility shift in response to CPT treatment. (B) Strains expressing the indicated catalytic domain mutants with GFP fused to the C-terminus were expressed in a chk1::ura4 disruption strain (NW158) from the chk1 promoter on the plasmid pSP1. Cells were visualized with a fluorescence microscope. Chk1+ is wild-type Chk1-GFP. Alleles of Chk1 are indicated by the mutated amino acid number and substitution. (C) cDNAs encoding wild-type Chk1 or Chk1S345A fused to GFP were expressed from the nmt1 promoter on the pREP1 vector under repressing conditions in a chk1::ura4 disruption strain (NW158). Live cells were visualized by fluorescence microscopy.

 


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  		Fig. 3. Serine 345 is phosphorylated and required for activation of Chk1 kinase activity in response to DNA damage. (A) A serine 345 phosphoantibody recognized Chk1 but not Chk1S345A after exposure to CPT. NW556 (Chk1) and NW492 (S345A) were grown to mid-log phase, treated with CPT and lysed. Lysate was incubated with F-7 anti-HA antibody, subjected to SDS-PAGE, and immunoblotted with S345 phosphoantibody or Y-11 anti-HA antibody. (B) Serine 345 phosphorylation is required for activation of Chk1 kinase activity in response to CPT. Chk1S345D is not constitutively activated. Immunoprecipitated Chk1, Chk1D155A, Chk1S345A and Chk1S345D were assayed for their ability to phosphorylate a peptide substrate. All values are from triplicate immunoprecipitations except Chk1+CPT and S345D+CPT, which are derived from two immunoprecipitations. (C) Full activation of Chk1 requires the checkpoint pathway protein Rad3. Chk1 was prepared as in B from a wild-type strain, NW223, and from a strain with a deletion of the rad3 protein, NW246, and assayed as above. All values are from triplicate immunoprecipitations; the data shown represents the mean, with error bars showing the standard deviation.

 


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  		Fig. 4. Mutation of serine 345 to alanine or aspartic acid compromises checkpoint function and viability following exposure to DNA damage. (A) NW228 (chk1+), NW107 (chk1::ura4), NW442 (chk1S345A) and NW477 (chk1S345D) cells harboring the cdc25-22 temperature-sensitive mutant were grown to mid-log phase at 25°C, plated on agar plates, shifted to 36.5°C for 2 hours, treated with UV light, shifted back to 25°C, and monitored for entry into mitosis. Symbols for strains are shown; closed symbols, + UV; open symbols, - UV. (B) NW223 (chk1+), NW158 (chk1::ura4), NW444 (chk1S345A) and NW463 (chk1S345D) cells were grown to mid-log phase, spread on agar plates, exposed to UV, and incubated for approximately 3 days. Colony number was determined and is expressed as a percentage of colonies appearing on untreated plates. Symbols for strains are as in A. (C) NW227 (chk1+), NW157 (chk1::ura4), NW443 (chk1S345A) and NW479 (chk1S345D) cells harboring the cdc17-K42 temperature-sensitive mutant were grown to mid-log phase, resuspended at 1x107 cells/ml, spotted onto agar plates at 10x dilutions, and incubated for approximately 3 days at 25°C, 32°C or 36°C. (D) Phosphorylation of Chk1 occurs in strains with the wee phenotype, which are dependent on chk1 for survival. Strains NW223 (wild-type), NW252 (mik1::ura4), NW253 (mik1::ura4 wee1-50), NW230 (wee1-50) and NW239 (rad1-1 wee1-50) were grown at 25°C and lysed for analysis by immunoblot with 12CA5 anti-HA antibody.

 


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  		Fig. 5. Mutation of serine 345 to alanine or aspartic acid abolishes association of Chk1 with 14-3-3 upon DNA damage. NW223 (Chk1), NW444 (S345A) and NW463 (S345D) were grown to mid-log phase, treated with CPT and lysed. Lysates were incubated with antibody against Rad 24 (UMDNJ 55), subjected to SDS-PAGE and immunoblotted with 12CA5 anti-HA antibody (IP). Control precipitations (NI) with non-immune rabbit serum were processed exactly as anti-Rad24 IPs. Samples of the lysates, before immunoprecipitation are shown in the left panel.

 

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