A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition.
The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+. Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.
Gould
K L
KL
Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Feoktistova
A
A
Fleig
U
U
D000705:Anaphase / Q000235:genetics*; D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D016153:Genes, Suppressor; D008677:Metaphase / Q000235:genetics*; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D010766:Phosphorylation; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine / Q000378:metabolism
D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; C577398:CUT9 protein, S pombe; D018797:Cell Cycle Proteins; D019926:Cyclin B; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; D014443:Tyrosine; D016203:CDC2 Protein Kinase