The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction |
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| Authors: | Margaux R. Audett Erin L. Johnson Jessica M. McGory Dylan M. Barcelos Evelin Oroszne Szalai Marcin R. Przewloka Thomas J. Maresca |
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| Affiliation: | University of North Carolina, Chapel Hill;aBiology Department, University of Massachusetts, Amherst, Amherst MA 01003;bMolecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, Amherst MA 01003;cInstitute for Life Sciences, School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK |
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| Abstract: | KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction. |
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