Student Theses and Dissertations

Date of Award

2012

Document Type

Thesis

RU Laboratory

Funabiki Laboratory

Abstract

Two regulatory mechanisms exist to ensure proper chromosome segregation in mitosis. First, improper kinetochore-microtubule attachments are destabilized through the error correction machinery. Second, the spindle assembly checkpoint (SAC) delays anaphase onset until all kinetochores have achieved bioriented microtubule attachments. Both of these mechanisms are mediated by several centromeric and kinetochore kinases, including Aurora B. Protein Phosphatase 1 (PP1) plays a counteracting role to Aurora B to stabilize kinetochore-microtubule attachments and silence the SAC. The regulation of PP1 to modulate these functions, however, remains enigmatic. Using the biochemical tools available in the Xenopus egg extract system, I show here that PP1 binds to the protein KNL1 (Spc105, Blinkin, CASC5) through an evolutionarily conserved RVxF motif. KNL1 is a member of the KMN network that forms the microtubule binding interface at the kinetochore. Using the genetic tools of Saccharomyces cerevisiae, I show that this interaction is essential for silencing the SAC, but has only a minimal effect on kinetochore-microtubule stability. Although phosphorylation of KNL1 by Aurora B can abrogate the KNL1- PP1 interaction, constitutive recruitment of PP1 by KNL1 is insufficient to prematurely silence the SAC. However, the amount of PP1 recruited to the kinetochore is tightly tuned, as targeting just one extra copy of PP1 to KNL1 is lethal. The data presented here leads to a model in which the KNL1-PP1 interaction acts to couple microtubule attachment with SAC signaling. Specific properties of the N-terminus of KNL1 may modulate this coupling, possibly though conformational changes upon microtubule attachment. In addition, there have been several other proteins found to recruit PP1 to the kinetochore, and how these regulatory subunits might cooperate to mediate the functions of PP1 will be discussed.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/535

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