Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function.
The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage. We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25. The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast. We identified a stretch of leucine residues as the domain in Chk1 that mediates the interaction with 14-3-3 proteins. Substitution of leucine residues with alanine disrupts the interaction with Rad24 and also prevents Chk1 from becoming phosphorylated in response to DNA damaging agents. Cells expressing the mutants are sensitive to UV radiation. In this study, we also show that Chk1 accumulates in the nucleus in response to DNA damage and this behavior is dependent on Rad24. Interestingly, the 14-3-3 binding domain mutants also fail to localize to the nucleus prompting a search for localization sequences within Chk1. Our investigations have identified the presence of both functional nuclear import and nuclear export sequences encoded in S. pombe Chk1 that, in conjunction with 14-3-3 proteins, may play a prominent role in regulating Chk1 localization and function.
Dunaway
Stephen
S
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Liu
Hsing-Yin
HY
Walworth
Nancy C
NC
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005455:Fluorescent Antibody Technique; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D009154:Mutation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029702:Schizosaccharomyces pombe Proteins; C501114:rad24 protein, S pombe; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D002166:Camptothecin