Sustained Mps 1 activity is required in mitosis to recruit O-Mad 2 to the Mad 1 – C-Mad 2 core complex

The Mps1 protein kinase plays an essential role in the spindle assembly checkpoint (SAC; Weiss and Winey, 1996), an inhibitory network that restrains anaphase until all kinetochores are stably attached to spindle microtubules (Musacchio and Salmon, 2007). Recent efforts have revealed that its catalytic activity is required for SAC function and chromosome alignment (Jelluma et al., 2008a,b; Tighe et al., 2008; Kwiatkowski et al., 2010; Sliedrecht et al., 2010; see Maciejowski et al. and Santaguida et al. in this issue). Exactly how Mps1 does this is unclear; although its SAC function is linked to kinetochore recruitment of Mad1 and Mad2 (Martin-Lluesma et al., 2002; Liu et al., 2003), Mps1 has also been implicated in aurora B regulation (Jelluma et al., 2008b), possibly explaining its role in chromosome alignment. Previously, when we used an RNAi complementation assay to inhibit Mps1’s catalytic activity, Mad2 was largely undetectable at kinetochores (Tighe et al., 2008). However, Mad1 recruitment was only partially affected, suggesting two possibilities: either Mps1 recruits Mad1 via a noncatalytic role or only low level Mps1 activity is required for Mad1 recruitment. Either way, because Mad1 directly recruits Mad2 to the kinetochore (Chung and Chen, 2002), the simplest explanation was that Mps1 activity promotes the Mad1–Mad2 interaction. This notion is at odds with current views of Mad2 function (Mapelli et al., 2007). The Mad2 template model posits that a Mad1 dimer acts as the kinetochore receptor for Mad2, which can adopt two conformations, open (O-Mad2) and closed (C-Mad2). When Mad2 binds Mad1, it adopts the closed conformation, forming the Mad1–C-Mad2 core complex. Because Mad2 can dimerize, this core complex in turn recruits two O-Mad2 molecules. These are handed over to Cdc20, and in doing so, Mad2 closes, generating two Cdc20–C-Mad2 complexes. Because the Mad1–C-Mad2 complex is very stable (Howell et al., 2004; Shah et al., 2004; Vink et al., 2006) and probably present in interphase where it binds the Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression. Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex