Practice question

Assembly of spindle assembly checkpoint proteins at kinetochores requires phosphorylation-dependent creation of docking sites on outer kinetochore scaffold. KNL1, also called Blinkin or Spc105, contains multiple MELT repeats (Met-Glu-Leu-Thr) that become phosphorylated when kinetochore is unattached. The kinase responsible is monopolar spindle 1, abbreviated Mps1, conserved from yeast to humans, dual-specificity kinase that localizes to unattached kinetochores via its TPR domain interacting with Ndc80 complex and calponin homology domains. Mps1 phosphorylation of MELT motifs generates binding platforms for Bub1-Bub3 complexes through Bub3 recognizing phospho-MELT. Bub1 then recruits Bub3-BubR1 and Mad1-Mad2, initiating MCC generation. Mps1 also phosphorylates Mad1 and other components to stabilize checkpoint signaling. Inhibition of Mps1 with small molecules like reversine results in checkpoint failure despite presence of unattached kinetochores, leading to rapid mitotic exit with missegregated chromosomes. Aurora A primarily regulates centrosome maturation, Cdc25 is phosphatase activating CDKs, ATR responds to DNA damage rather than kinetochore attachment. Live-cell imaging shows Mps1 recruitment is dynamic, peaking in early prometaphase and declining as kinetochores attach, with phosphatase PP2A-B56 opposing its activity to prevent excessive checkpoint signaling, ensuring graded response proportional to number of unattached kinetochores and integrating microtubule occupancy with intra-kinetochore tension measurements.