Mad3/BubR1 phosphorylation during spindle checkpoint activation depends on both Polo and Aurora kinases in budding yeast.
During mitosis the spindle assembly checkpoint (SAC) delays the onset of anaphase and mitotic exit until all chromosomes are bipolarly attached to spindle fibers. Both lack of attachment due to spindle/kinetochore defects and lack of tension across kinetochores generate the "wait anaphase" signal transmitted by the SAC, which involves the evolutionarily conserved Mad1, Mad2, Mad3/BubR1, Bub1, Bub3 and Mps1 proteins, and inhibits the activity of the ubiquitin ligase Cdc20/APC, that promotes both sister chromatid dissociation in anaphase and mitotic exit. In particular, Mad3/BubR1 is directly implicated, together with Mad2, in Cdc20 inactivation in both human and yeast cells, suggesting that its activity is likely finely regulated. We show that budding yeast Mad3, like its human orthologue BubR1, is a phosphoprotein that is hyperphosphorylated during mitosis and when SAC activation is triggered by microtubule depolymerizing agents, kinetochore defects or lack of kinetochore tension. In vivo Mad3 phosphorylation depends on the Polo kinase Cdc5 and, to a minor extent, the Aurora B kinase Ipl1. Accordingly, replacing with alanines five serine residues belonging to Polo kinase-dependent putative phosphorylation sites dramatically reduces Mad3 phosphorylation, suggesting that Mad3 is likely an in vivo target of Cdc5.
Rancati
Giulia
G
Dipartimento di Biotecnologie e Bioscienze, Universita' degli Studi di Milano-Bicocca, Milan, Italy.
Crispo
Valentina
V
Lucchini
Giovanna
G
Piatti
Simonetta
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D064127:Aurora Kinases; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D018386:Kinetochores / Q000378:metabolism; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D004718:Saccharomycetales / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D011494:Protein Kinases; D064127:Aurora Kinases; C576961:IPL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D018844:Cyclin-Dependent Kinases; D015739:Nocodazole