Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling.
The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N-terminal domain of Ndc80 (Ndc80(1-257)), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc80(1-257) in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild-type-like bipolar kinetochore-microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1-dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores.
Kemmler
Stefan
S
Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg, Germany.
Stach
Manuel
M
Knapp
Maria
M
Ortiz
Jennifer
J
Pfannstiel
Jens
J
Ruppert
Thomas
T
Lechner
Johannes
J
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D064127:Aurora Kinases; D002453:Cell Cycle / Q000502:physiology*; D018816:Genes, cdc; D047908:Intracellular Signaling Peptides and Proteins; D018386:Kinetochores / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D008941:Spindle Apparatus / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D064127:Aurora Kinases; C576961:IPL1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C093416:MPS1 protein, S cerevisiae; D000409:Alanine