The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe.
In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.
Ozoe
Fumiyo
F
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan.
Kurokawa
Rumi
R
Kobayashi
Yasuyo
Y
Jeong
Hee Tae
HT
Tanaka
Katsunori
K
Sen
Kikuo
K
Nakagawa
Tsuyoshi
T
Matsuda
Hideyuki
H
Kawamukai
Makoto
M
D048948:14-3-3 Proteins; D001692:Biological Transport; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015825:Chromosomes, Fungal; D003470:Culture Media; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D003470:Culture Media; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C501114:rad24 protein, S pombe; C489574:rad25 protein, S pombe; C071596:ste11 protein, S pombe; D014446:Tyrosine 3-Monooxygenase; D020928:Mitogen-Activated Protein Kinases; C077892:BYR2 protein, S pombe; D020930:MAP Kinase Kinase Kinases; D004265:DNA Helicases; C492657:Ras1 protein, S pombe; D018631:ras Proteins