ORIGINAL ARTICLE The Arabidopsis 14-3-3 protein, GF14x, binds to the Schizosaccharomyces pombe Cdc25 phosphatase and rescues checkpoint defects in the rad24 ) mutant Received: 21 February 2003 / Accepted: 24 June 2003 / Published online: 26 August 2003 Ó Springer-Verlag 2003 Abstract The fission yeast (S. pombe) mitotic inducer gene, Spcdc25, interacts with the plant cell cycle to establish a small cell size phenotype compared with wild- type cells. We have investigated the nature of this interaction by yeast two-hybrid screening using Spcdc25 as bait in a cDNA library prepared from root tips of Arabidopsis thaliana (L.) Heynh. Three 14-3-3 proteins were detected: G-box Factor-like (GF)14j, k and x; binding with Spcdc25 was confirmed by an independent immunoprecipitation assay. To test for cell cycle checkpoint function, GF14j, k and x were transformed independently, using the strong nmt1 + promoter, into rad24 ) , a fission yeast mutant deficient in a 14-3-3 checkpoint protein. When exposed to UV irradiation or in the presence of 10 mM hydroxyurea, only cells transformed with GF14x could fully rescue the defects in the DNA-damage and DNA-replication checkpoints of this mutant. Supporting evidence for a GF14x cell cycle function was provided by semi-quantitative reverse transcription–polymerase chain reaction indicating that expression of this gene was elevated in regions of the plant that comprise dividing cells whereas GF14j and k expression was more evenly detected in all tissues examined. The data are consistent with the hypothesis that interaction between Spcdc25 and the plant cell cycle occurs at the level of a 14-3-3 protein with distinct checkpoint properties. Keywords Arabidopsis (cell cycle) Æ Cell cycle checkpoints Æ Schizosaccharomyces Æ Saccharomyces Æ 14-3-3 proteins (GF14k, j, and x) Abbreviations ATM: ataxia telangiectasia-mutated gene Æ CDK: cyclin-dependent kinase Æ GF14: G-box Factor-like 14-3-3 protein Æ Rad: radiation sensitive Æ RT–PCR: reverse transcription–polymerase chain reaction Æ UV: ultraviolet Introduction In the eukaryote cell cycle, chromosomes separate at mitosis and are replicated in S-phase, events that in most proliferative cells are temporally separated by G1 and G2. The cell cycle is regulated by a series of cyclin-dependent protein kinases (CDKs) that drive cells through specific transitions, notably at G1/S and G2/M (Morgan 1997). In fission yeast, one CDK, Cdc2, functions at both transi- tions (Nurse 1990). In higher eukaryotes there are several CDKs that, in partnership with different cell cycle-specific cyclins, function at different stages of the cell cycle (Morgan 1997). In animals, CDK1 binds with cyclinB and kinase activity peaks at the G2/M transition (Dunphy 1994) whereas in Arabidopsis at least two classes of CDK function at this transition: CDC2A and CDC2B, al- though in plants, the exact partner cyclin is not known for all CDKs (Stals et al. 2000). At G2/M, dephosphorylation of the CDK occurs at tyrosine15 in fission yeast (Gould and Nurse 1989) and also at threonine14 in animals (Dunphy 1994). Cdc25 phosphatase is responsible for this Planta (2003) 218: 50–57 DOI 10.1007/s00425-003-1083-7 David A. Sorrell Æ Angela M. Marchbank David A. Chrimes Æ J. Richard Dickinson Hilary J. Rogers Æ Dennis Francis Claire S. Grierson Æ Nigel G. Halford D. A. Chrimes Æ J. R. Dickinson H. J. Rogers Æ D. Francis (&) School of Biosciences, Cardiff University, PO Box 915, Cardiff, CF10 3TL, UK E-mail: francisd@cardiff.ac.uk Fax: +44-29-20874305 C. S. Grierson School of Biological Sciences, Bristol University, Woodland Road, Bristol, BS8 1UG, UK N. G. Halford Department of Crop Performance and Improvement, Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK D. A. Sorrell Hannah Research Institute, Ayr, KA6 5HL, UK A. M. Marchbank Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK