MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

Abstract

Cyclin B–dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

Triggering mitosis

“…Specific enrichment of Cdk1 kinase activity at a particular cellular localisation could also be important. Accordingly, cyclin B is known to be recruited to the kinetochore and has recently been shown to contribute to spindle assembly checkpoint signalling by phosphorylating Mps1 . A large subset of the cyclin B‐specific substrates that we have identified using degron‐mediated depletion is indeed associated with the centromere/kinetochore, supporting the importance of this targeted localisation of cyclin B .…”

Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

“…As a consequence, the length of the mitotic arrest was impaired. In keeping with these findings, the spindle checkpoint was strongly compromised in cyclin B1‐depleted cells . These observations support the idea that the cyclin B1‐MAD1 interaction creates a positive feedback loop because the local concentration of MAD1‐bound CDK1‐cyclin B1 at an unattached kinetochore allows for more efficient phosphorylation and kinetochore localization of MPS1, in turn recruiting more MAD1.…”

“…The multiple CDK1‐cyclin B1 targets within the SAC discussed above highlight that CDK1‐cyclin B1 is more intimately involved in the regulation of the SAC than a superficial analysis of SAC wiring may suggest. A further demonstration of the close relationship of CDK1‐cyclin B1 with its substrates in the mitotic checkpoint signalling cascade is the finding that cyclin B1 itself is localized to unattached kinetochores and displaced from these upon microtubule binding, just like a bona fide SAC protein (Fig. ).…”

“…In mitosis, APC/C is thus associated with two functionally distinct molecules of CDC20. B1 failed to localize to unattached kinetochores in MAD1 depleted cells, suggesting that MAD1 is the main MPS1 regulated kinetochore receptor for cyclin B1 [117,120]. Further analysis showed that cyclin B1 is bound to the kinetochore via the first 100 amino acids of the N-terminus of MAD1.…”

“…Complementary proteomic and cell biological analysis demonstrated that MAD1 is one of the major components of cyclin B1 complexes in mitosis. Indeed, cyclin B1 failed to localize to unattached kinetochores in MAD1 depleted cells, suggesting that MAD1 is the main MPS1 regulated kinetochore receptor for cyclin B1 . Further analysis showed that cyclin B1 is bound to the kinetochore via the first 100 amino acids of the N‐terminus of MAD1.…”