At this point, we do not know how exactly a PHLPP is recruited to the Akt-SRPK1 complex

or pH3T3 provided by Survivin. Borealin1-221 contains all the Cdk1 phosphorylation sites required to bind to Sgo1/2. In principle, a CPC monomer created with Borealin1-221 might simultaneously bind pH3T3 via Survivin and pH2AT120 via Sgo1/2. Our data argue that if this is the case, the contribution of pH2AT120 to the binding energy must be minimal. Otherwise, Borealin1-221 would show significantly slower exchange than Borealin1-110. Borealin1-110 and Borealin1-221 could partially replace endogenous Borealin in supporting progression through mitosis. This partial rescue suggests that slow exchange of the CPC at the centromere is required to properly coordinate chromosome attachment to the spindle. Rapid exchange is also consistent with the ability of truncated Borealin proteins to partially displace INCENP from the centromere. We hypothesized that these effects are the consequence of forming a monomeric CPC that presents half the number of binding sites for centromeric receptors, which spends more time in the cytoplasm with a resulting partial loss of function. To investigate this idea, the dimerization domain of Borealin was replaced with FKBP allowing inducible dimerization 28,36,37. Borealin1-221-FKBP was localized to centromeres and the cytoplasm in the absence of the AP20187 dimerizer. These conditions also dispersed the CPC from the centromere. Addition of AP20187 rescued centromere localization of both Borealin1-221-FKBP and CPC showing that dimerization is required for efficient association with the centromere. The Borealin1-110-FKBP fusion was difficult to detect at centromeres in the absence of AP20187, but still able to displace the CPC suggesting that the N-terminal triple helical bundle is intact. AP20187 rescued Borealin1-110-FKBP, Aurora B, INCENP and Survivin providing more evidence that the central region of Borealin plays a minor role in inner centromere CPC targeting. In contrast, LY3039478 Borealin110+D.D displayed similar kinetics compared to fragments containing amino acids 1-110 or 1-221. We hypothesize that the two CPC arms must be a minimum distance to support dual interaction with the centromere. We used several approaches to correlate CPC localization and the known centromere receptors, pH3T3 and pH2AT120. For example, Mps1 inhibition reduced pH2AT120 by ~92%, but did not affect pH3T3 nor CPC localization or dynamics. CPC localization and Borealin dynamics were also unchanged in mitotic cells exposed to ZM447439 which reduced pH2AT120 by ~92% and pH3T3 by ~72%. Therefore, CPC localization can be uncoupled from the majority of pH2AT120 and pH3T3 if the inhibitors are added after cells enter mitosis. 5Itu was more efficient than either ZM447439 or reversine at reducing pH3T3 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Commun. Author manuscript; available in PMC 2015 October 09. Bekier et al. Page 9 and subsequently reduced all CPC family members at centromeres and increased Borealin centromere dynamics. Detailed confocal analysis of 5Itu-treated cells revealed a subpopulation of Borealin proximal to kinetochores. Analysis of Borealin-FKBP fusions showed that localization near the kinetochore only occurs when the CPC is dimerized and only when the central domain of Borealin implicated in pH2AT120 recognition is intact. We interpret this observation to indicate that a CPC dimer, but not monomer associates with two copies of a kinetochore-proximal target which may be pH2AT120. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850718,22102576 Presumably the mo