First published online 7 December 2004
doi: 10.1242/jcs.01570
Journal of Cell Science 118, 39-50 (2005)
Published by The Company of Biologists 2005
Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function
Stephen Dunaway1,2,
Hsing-Yin Liu1,2 and
Nancy C. Walworth1,2,3,*
1 Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
2 Joint Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Graduate School of Biomedical Sciences and Rutgers University, Piscataway, NJ 08854, USA
3 Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
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Fig. 1. Identification of residues in Chk1 required for the interaction with Rad24. (A) Domain structure of Chk1 highlighting the region required for interaction between Chk1 and Rad24 (amino acids 286-319). (B) Result of yeast two-hybrid ß-galactosidase assay. Strains transformed with the pACT/Rad24 plasmid and the indicated alleles of chk1 in the pAS2/Chk1 plasmid were exposed to X-Gal. Only the wild-type allele and the kinase domain mutant K38A show expression of the lacZ gene as revealed by X-gal reactivity. (C) Western blot of lysates from cells expressing the indicated alleles of Chk1 that had (+) or had not (–) been exposed to the topoisomerase-I poison camptothecin (CPT). Chk1 is detected with anti-HA antibody to detect the tag on the C-terminus of Chk1. Only the wild-type protein undergoes the mobility shift representative of Chk1 phosphorylation. (D) Immunoprecipitation of lysates shown in C to evaluate the interaction of alleles of Chk1 with Rad24. Immunoprecipitations were carried out with antibodies against Rad24 or with a non-immune antibody. The immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose and probed by immunoblot with antibody against the HA tag on the C-terminus of Chk1.
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Fig. 2. chk1L A mutants exhibit a loss-of-function phenotype. (A) UV survival was monitored for the indicated strains by a colony survival assay after exposure to 40, 80 or 160 J/m2 UV. Triplicate plates were prepared and the data represent the mean survival with error bars indicating the standard deviation. (B) Cells from the indicated strains were plated on YEA plates and incubated at 25°C, 32°C and 36°C to assay for synthetic lethality in a DNA ligase-deficient background cdc17-K42. (C) The indicated chk1 alleles were crossed into a cdc25-22 background in order to allow synchronization. G2-blocked cells were exposed to UV and released at 25°C. Cells were then scored for passage through mitosis (see Materials and Methods).
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Fig. 3. Chk1 protein accumulates in the nucleus after DNA damage. (A) A strain with integrated Chk1:HA was examined by immunofluorescence with anti-HA antibody. Cultures grown to mid-log phase were treated with 40 µM CPT for 2.5 hours to generate DNA damage, then fixed and processed for immunofluorescence. Antibody to a cytoplasmic protein Ded1 is also shown. Bar, 10 µm. (B) Quantification of immunofluorescence signal was performed by measuring pixel intensity in the nuclei and in an equivalent area of the cytoplasm. Nuclear to cytoplasmic ratios of these signals were then generated and cells were sorted into the three categories shown. (C) Quantification was performed and is presented for the integrated wild-type chk1:HA allele (an average of two independent experiments is shown). (D) Quantification is presented on a similar immunofluorescence experiment performed with a strain deleted for endogenous chk1 and instead expressing Chk1:HA from the pSP1 plasmid (an average of four independent experiments is shown).
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Fig. 4. Chk1 accumulation in the nucleus is Rad24 dependent. Chk1 immunofluorescence was performed in a rad24 strain after treatment for 2.5 hours with or without 40 µM CPT. Cells were also stained with DAPI. Bar, 10 µm.
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Fig. 5. Chk1 contains a functional NLS. (A) A strain expressing the chk1-4A mutant integrated at the chk1 locus was treated with 40 µM CPT and processed for immunofluorescence. (B) UV survival was measured for the indicated strains after exposure to 50, 100, 150 and 200 J/m2 UV. UV survival experiments were conducted in triplicate. (C) Western blotting was performed on protein lysates from the indicated strains that were treated with or without 40 µM CPT for 2.5 hours. Chk1 mobility shift was monitored after SDS-PAGE using anti-HA antibody. (D) A strain expressing the chk1-4A + SV40 NLS was treated with 40 µM CPT and processed for immunofluorescence. (E) UV survival was measured for the indicated strains as in B. (F) Addition of the SV40-NLS to wild-type Chk1 enhances nuclear localization as detected by immunofluorescence. Bars, 10 µm.
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Fig. 6. chk1LA mutants display the same phenotypes as a chk1– strain. (A-D) Chk1 immunofluorescence was performed on strains expressing the indicated plasmid-borne Chk1 mutants after treatment with or without 40 µM CPT for 2.5 hours. Quantification was then performed (an average of two independent experiments is shown). Bar, 10 µm. (E) UV survival was monitored for the indicated strains by colony counting after exposure to 40, 80 or 160 J/m2 UV. (F) Western blotting was performed with 12CA5 antibody on protein lysates from the indicated strains treated with or without 40 µM CPT for 2.5 hours.
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Fig. 7. Chk1 contains a functional NES. (A-D) Chk1 immunofluorescence was performed on strains harboring the indicated plasmid-borne Chk1 alleles after treatment with or without 100 ng/ml LMB for 1-2 hours. Quantification was then performed (an average of four independent experiments is shown for the wild-type, three independent experiments for each of the mutants). Bar, 10 µm.
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Fig. 8. Model for the role of localization in regulation of Chk1 in fission yeast. When Chk1 binds Rad24 the 14-3-3 protein may act to open up the conformation of Chk1 to expose an NLS, while at the same time blocking access of Crm1 to the NES. This would allow Chk1 to enter the nucleus. Subsequent dissociation of 14-3-3 would expose the NES permitting Crm1 to bind and mediate export. When cells incur DNA damage, Chk1 becomes phosphorylated and its interaction with 14-3-3 protein is stimulated approximately tenfold (Chen et al., 1999 ). Nuclear accumulation of Chk1 would thus result from the stabilized interaction of Chk1 with Rad24 and the consequent reduction in its association with Crm1.
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© The Company of Biologists Ltd 2005
