. We show that this responsiveness arises because the SAC primes kinetochore phosphatases to induce negative feedback and silence its own signal. Active SAC signalling recruits PP2A-B56 to kinetochores where it antagonises Aurora B to promote PP1 recruitment. PP1 in turn silences the SAC and delocalises PP2A-B56. Preventing or bypassing key regulatory steps demonstrates that this spatiotemporal control of phosphatase feedback underlies rapid signal switching at the kinetochore by; 1) allowing the SAC to quickly transition to the ON state in the absence of antagonising phosphatase activity, and 2) ensuring phosphatases are then primed to rapidly switch the SAC signal OFF when kinetochore kinase activities are diminished by force-producing microtubule attachments.The spindle assembly checkpoint (SAC) is globally activated at mitotic entry and only extinguished when all kinetochores have established force-producing microtubule attachments 2,3 . At each individual kinetochore however, the SAC responses are much more dynamic. Here, localised SAC signalling switches rapidly between the ON and OFF states depending on microtubule occupancy [2][3][4][5][6] .Exactly how kinetochores manage to achieve this rapid signal switching remains unknown. To address this we initially focussed on characterising the kinetochore phosphatases responsible for SAC silencing in mammalian cells. We performed a targeted screen with siRNAs to 222 individual phosphatase subunits to identify those that regulate mitotic exit in mammalian cells. 48 hours after siRNA transfection, cells were synchronised in mitosis using the microtubule poison nocodazole, after which mitotic exit was forced by the small molecule MPS1 inhibitor reversine 7 for 1 hour. (Fig.1b). Live monitoring of endogenous Cyclin B1 levels 16 showed that B56 depletion prevented efficient APC/C activation following MPS1 inhibition (Fig.1c). This indicated that PP2A-B56 depletion did not simply delay mitotic exit, but in fact prevented SAC silencing. PP2A-B56 has recently been shown to localise to the outer kinetochore via interaction with a short phosphorylated motif in BUBR1 (termed KARD) [17][18][19] . We found that all B56 isoforms that we tested (B56, ,  1 ,  3 , ) localised to the centromere/kinetochores regions of mitotic chromosomes, with some more clearly enriched on kinetochores than others (B56 1 ,  3 , ; Supplementary Fig.1a). We next deleted the B56 binding motif from BUBR1 (BUBR1 ∆KARD ; Supplementary Fig.1b-d), which specifically abolished B56 kinetochore localisation (Fig.1d, e and supplementary fig.1e), and delayed mitotic exit following MPS1 inhibition with either reversine (Fig.1f) or the distinct inhibitor ( Supplementary Fig.1f). These delays were accentuated by concomitant B56 depletion, which even allowed cells to mount a prolonged arrest with a high dose of reversine or . This was unrelated to effects on centromeric PP2A-B56 because SGO1 depletion caused mitotic arrest due to reduced centromeric PP2A and loss of sister chromatid cohesion, as expe...

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.heart ͉ oxidative stress ͉ myocardium ͉ redox signaling