The Phosphatase PP1 Promotes Mitotic Slippage Through Mad3 Dephosphorylation

Abstract

Accurate chromosome segregation in mitosis requires the bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the Spindle Assembly Checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays the onset of anaphase until all chromosomes are bipolarly attached. SAC signalling fires at kinetochores and involves the formation of a soluble Mitotic Checkpoint Complex (MCC) made by Mad2, Mad3/BubR1, Bub3 and Cdc20, which inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex (APC). The mitotic delay imposed by SAC, however, is not everlasting. After a variable time during which kinetochores have failed to establish bipolar connections, cells escape from the SAC-induced mitotic arrest through a process called mitotic slippage. Mitotic slippage occurs in the presence of SAC signalling at kinetochores, but whether and how MCC stability and APC inhibition are actively controlled during slippage is not known. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached. PP1 turns off SAC signalling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores. Here we show that in budding yeast PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilises the MCC, but in spite of that, does not affect the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.