Accurate chromosome segregation is essential for cell viability, organismal development, and tumor suppression. Accordingly, eukaryotes have evolved several mechanisms to defend against chromosome segregation errors. Paramount among these is the so-called spindle assembly checkpoint (SAC), which inhibits anaphase onset until all kinetochore pairs have attached to microtubules (MTs) emanating from both spindle poles, generating a stable configuration termed chromosome biorientation (for review see Musacchio and Salmon, 2007
). In biochemical terms, the SAC acts by inhibiting the Cdc20-bound form of the anaphase-promoting complex/cyclosome (APC/C), a large ubiquitin protein ligase (Peters, 2006
). Repression of APC/CCdc20
activity stabilizes its downstream targets including securin and cyclin B, which directly block sister chromatid separation and mitotic exit.
Early time-lapse and laser ablation studies pointed to a central role for unattached kinetochores in checkpoint signaling (Rieder et al., 1994
; Li and Nicklas, 1995
). Consistent with this notion, all known SAC transducers, including the protein kinases Mps1, Bub1, and BubR1 and the nonkinase components Mad1, Mad2, and Bub3, associate with unattached kinetochores in prometaphase (for review see Musacchio and Salmon, 2007
). In particular, it is thought that kinetochore-localized Mad1/Mad2 heterodimers catalyze the conversion of soluble open Mad2 (O-Mad2) to a closed conformer (C-Mad2) that stably binds to and inhibits Cdc20 (De Antoni et al., 2005
). However, other compelling data argue that SAC signaling does not entirely depend on kinetochores. First, complexes of Cdc20 bound to Mad2 and/or BubR1 (sometimes referred to as the mitotic checkpoint complex) have been detected in interphase mammalian cells and yeast strains that lack functional kinetochores (Fraschini et al., 2001
; Sudakin et al., 2001
). Second, Mad2 and BubR1 are required not only to prolong mitosis in the presence of unattached kinetochores, but also to specify the minimum length of M phase under unperturbed conditions (Meraldi et al., 2004
). In contrast, inactivation of other SAC components or factors required for kinetochore–MT binding does not accelerate M phase. Third, BubR1’s essential mitotic functions can be reconstituted with an N-terminal fragment that binds Cdc20 but cannot localize to kinetochores (Malureanu et al., 2009
). Together, these observations argue that Mad2 and BubR1 are components of a cytosolic timer that actively restrains anaphase onset, affording early mitotic cells time to mature their kinetochores and (if necessary) engage the kinetochore-dependent branch of the SAC. However, the upstream factors that govern this timer remain elusive.
Although the SAC is conserved throughout Eukarya, efforts to define the order in which its components act relative to one another have yielded unexpectedly divergent results. For instance, studies in human cells have consistently positioned Mps1 near the distal end of the SAC, as depleting this kinase via RNAi results in the selective loss of Mad2 from kinetochores (Stucke et al., 2002
; Liu et al., 2003
; Jelluma et al., 2008
; Tighe et al., 2008
). In contrast, genetic analyses in yeast and immunodepletion experiments in Xenopus laevis
egg extracts place Mps1 at the apex of the SAC, upstream of not only Mad2 but also Bub1, BubR1/Mad3, and Mad1 (Hardwick et al., 1996
; Abrieu et al., 2001
; Vigneron et al., 2004
; Wong and Fang, 2005
). Similarly, although human Mps1 reportedly facilitates chromosome alignment by direct phosphorylation of the aurora B kinase regulator borealin, and thus is necessary to sustain full aurora B activity in human cells (Jelluma et al., 2008
), the aurora B–related kinase Ipl1 retains its normal localization and full activity in Mps1-deficient yeast strains (Maure et al., 2007
). These findings have been interpreted as evidence of species-specific differences in kinetochore organization and SAC regulation, but other explanations (e.g., technical issues related to the completeness or specificity of Mps1 inactivation) have not been excluded.
To clarify these issues, we created human cells in which both copies of the MPS1 locus could be deleted via gene targeting. The resulting MPS1-null cells were complemented with versions of the kinase that differ at a single amino acid within the ATP-binding site, conferring resistance or sensitivity to bulky purine analogues. Using this chemical genetic system, we investigated the role of Mps1 in M phase progression and SAC signaling. Our experiments identify a novel interphase function for Mps1, whereby it ensures that Cdc20 binds Mad2 and BubR1 before kinetochores have matured and can generate their own anaphase inhibitory signals. Mps1 is also critical for the subsequent phase of SAC signaling, as its inhibition evicts all known SAC mediators from prometaphase kinetochores. Furthermore, we find that although human Mps1 indeed controls chromosome biorientation, it does so independently of aurora B regulation, as indicated by undiminished phosphorylation of multiple aurora B substrates in Mps1-inhibited cells. Collectively, these findings reveal new insights into the SAC response in mammalian cells and provide new tools for interrogating this response in a rapid and specific manner without collateral inhibition of aurora B.