Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28.
In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.
Institute of Molecular and Cell Biology, National University of Singapore.
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D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins; D019926:Cyclin B; D016213:Cyclins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D019000:Phosphotyrosine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D006656:Protamine Kinase / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure; D014158:Transcription, Genetic
C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D016213:Cyclins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019000:Phosphotyrosine; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D006656:Protamine Kinase; D017489:CDC28 Protein Kinase, S cerevisiae