KNL1/Spc105 Recruits PP1 to Silence the Spindle Assembly Checkpoint

Abstract

SUMMARY The spindle assembly checkpoint (SAC) delays anaphase onset until kinetochores accomplish bioriented microtubule attachments [1]. While several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2–4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1 (PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of this interaction is dispensable. However, the amount of PP1 targeted to kinetochores must be finely tuned, since recruitment of either none or one extra copy of PP1 to Spc105 is detrimental, illustrating the vital impact of targeting an exiguous fraction of PP1 to the kinetochore. We propose that the PP1-Spc105 interaction enables local regulation of dynamic phosphorylation and dephosphorylation at the kinetochore to couple microtubule attachment and SAC silencing.

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…PP1 reverses the Mps1-mediated phosphorylation on Spc105, and it likely has additional key substrates (Pinsky et al 2009;London et al 2012). Spc105 may be involved in a checkpoint feedback loop because it has been implicated as the kinetochore receptor for PP1 in budding and fission yeast (Meadows et al 2011;Rosenberg et al 2011). However, direct binding between the budding yeast Spc105 and PP1 proteins was not demonstrated in budding yeast (Rosenberg et al 2011), and the Fin1 kinetochore protein also binds to PP1 (Akiyoshi et al 2009b).…”

“…In addition to contributing to KMN function, Spc105 also appears to be a scaffold for other outer kinetochore proteins. It recruits the Bub1 and Bub3 proteins to the kinetochore, and it may be a regulatory subunit for PP1 at the kinetochore (Kiyomitsu et al 2007(Kiyomitsu et al , 2011Liu et al 2010;Rosenberg et al 2011) (discussed below, The Spindle Checkpoint). The Ndc80 complex has two globular head domains that are connected by a long rod (Wei et al 2005;Ciferri et al 2008).…”

“…Spc105 may be involved in a checkpoint feedback loop because it has been implicated as the kinetochore receptor for PP1 in budding and fission yeast (Meadows et al 2011;Rosenberg et al 2011). However, direct binding between the budding yeast Spc105 and PP1 proteins was not demonstrated in budding yeast (Rosenberg et al 2011), and the Fin1 kinetochore protein also binds to PP1 (Akiyoshi et al 2009b). It is therefore still unclear precisely how PP1 is recruited and regulated to silence the checkpoint in yeast.…”

The Renaissance or the cuckoo clock

“…As a result, the closer the kinetochore is to the centromere, the higher the effective activity of Aurora B. This has led to the following model for microtubule capture and error correction, although it should be noted that the recruitment of PP1 to KNL1 may have an equally, or more, important role in the SAC [66] (see below). When a microtubule is first captured by the kinetochore, it enters a region of high Aurora B activity.…”

“…Evidence from studies on budding and fission yeast [66,88 -91], and recently on human cells [92], indicates that Aurora B has an important role in the SAC, and that this role again depends on the antagonism between Aurora B and PP1. More specifically, the recruitment of PP1g to KNL1 (or Spc7 in fission yeast) at kinetochores is required to silence the SAC [66,90,93]. Moreover, as Aurora B prevents PP1g binding to kinetochores [65], this means that the SAC can only be silenced when kinetochores are pushed away from the centromeres by microtubules, thereby coupling the SAC to error correction.…”