Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.
The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
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D016203:CDC2 Protein Kinase / Q000235:genetics*; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D005656:Fungal Proteins / Q000235:genetics*; D005816:Genetic Complementation Test; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine / Q000737:chemistry*
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; C102333:histone H1 kinase; C090679:mik1 protein, S pombe; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase