N the kinetochore and, surprisingly, the bulk pool is at the

N the kinetochore and, surprisingly, the bulk pool is at the inner kinetochore/centromere. Moreover, Plk1 operates at chromatin and in the inner centromere in a manner that is distinct and separable from Plk1’s 17 19 role in stabilizing microtubule attachments at the outer kinetochore . The separability of Plk1 operation within the kinetochore provides an opportunity to match functional and phosphoproteomic signatures, and to identify phosphorylation events that regulate specific aspects of mitotic progression. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Diffusible Plk1 fails to function at the kinetochore To interrogate Plk1 signaling at the kinetochore, we employed a chemical genetic system wherein each of two distinct Plk1 alleles is separately controlled by chemical inhibitors. Briefly, human RPE1 cells had the endogenous PLK1 exon 3 deleted and rescued by PD-1/PD-L1 inhibitor 2 chemical information Aglafoline web GFPPlk1as, which encodes chemical sensitivity to 3-methylbenzyl-pyrazolopyrimidine through two point mutations. When wild type Plk1 is co-expressed in cells with Plk1as, these complement, and either allele can execute the essential functions of this kinase. However, Plk1wt and Plk1as are separately controlled: Plk1wt is sensitive to the pharmacologic inhibitor, BI-2536 and resistant to 3-MB-PP1, whereas Plk1as is resistant to BI-253633. This empowers exquisite spatial and temporal control of Plk1 activity along the kinetochore-centromere axis. Plk1 signaling at the kinetochore might not require localization to exert its functions. To test this, we generated lines that stably co-express GFP-Plk1as and either Plk1wt, or Plk1aa, which has a wild type kinase domain but is delocalized by virtue of a mutant PBD. The PBD of Plk1aa has two point mutations –sufficient to disrupt Plk1 8 14 localization,. For the delocalized construct, we used either N-terminal Flag tag or mCherry tag . Both are expressed and exhibit catalytic activity similar to Plk1wt. As expected, neither localize to kinetochores, centrosomes, or the spindle midzone, yet both partially restore 10 12 bipolar spindles, consistent with previous findings,. We previously titrated low inhibitor concentrations to observe Plk1 loss-of-function 12 phenotypes in which a bipolar spindle is preserved. Under these conditions chromosomes fail to align at the midline, presumably from impaired kinetochore function. As expected, Plk1wt restored chromosome alignment . With Plk1aa or Ch-Plk1aa, however, chromosome alignment remained impaired. Thus, a functional PBD is required for Plk1 to align mitotic 10 34 chromosomes, consistent with earlier reports,. Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 4 A second loss-of-function phenotype of Plk1 is lagging anaphase chromosomes, which occur at very modest losses of Plk1 activity after chromosome alignment on a bipolar 12 spindle and silencing of the mitotic checkpoint. We tested if delocalized Plk1 was sufficient to restore accurate anaphase chromosome segregation. As expected, Plk1wt localized and restored chromosome segregation compared to vector cells, yet both Plk1aa and Ch-Plk1aa failed to do so . We conclude that PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19856273 PBD-dependent kinase localization is required for both chromosome alignment and segregation. Recapitulating Plk1 at the kinetochore We next considered how Plk1 localization within the kinetochore might control function. At the extremes, Plk1 could bind each substrate directly or it could bind a single p.N the kinetochore and, surprisingly, the bulk pool is at the inner kinetochore/centromere. Moreover, Plk1 operates at chromatin and in the inner centromere in a manner that is distinct and separable from Plk1’s 17 19 role in stabilizing microtubule attachments at the outer kinetochore . The separability of Plk1 operation within the kinetochore provides an opportunity to match functional and phosphoproteomic signatures, and to identify phosphorylation events that regulate specific aspects of mitotic progression. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Results Diffusible Plk1 fails to function at the kinetochore To interrogate Plk1 signaling at the kinetochore, we employed a chemical genetic system wherein each of two distinct Plk1 alleles is separately controlled by chemical inhibitors. Briefly, human RPE1 cells had the endogenous PLK1 exon 3 deleted and rescued by GFPPlk1as, which encodes chemical sensitivity to 3-methylbenzyl-pyrazolopyrimidine through two point mutations. When wild type Plk1 is co-expressed in cells with Plk1as, these complement, and either allele can execute the essential functions of this kinase. However, Plk1wt and Plk1as are separately controlled: Plk1wt is sensitive to the pharmacologic inhibitor, BI-2536 and resistant to 3-MB-PP1, whereas Plk1as is resistant to BI-253633. This empowers exquisite spatial and temporal control of Plk1 activity along the kinetochore-centromere axis. Plk1 signaling at the kinetochore might not require localization to exert its functions. To test this, we generated lines that stably co-express GFP-Plk1as and either Plk1wt, or Plk1aa, which has a wild type kinase domain but is delocalized by virtue of a mutant PBD. The PBD of Plk1aa has two point mutations –sufficient to disrupt Plk1 8 14 localization,. For the delocalized construct, we used either N-terminal Flag tag or mCherry tag . Both are expressed and exhibit catalytic activity similar to Plk1wt. As expected, neither localize to kinetochores, centrosomes, or the spindle midzone, yet both partially restore 10 12 bipolar spindles, consistent with previous findings,. We previously titrated low inhibitor concentrations to observe Plk1 loss-of-function 12 phenotypes in which a bipolar spindle is preserved. Under these conditions chromosomes fail to align at the midline, presumably from impaired kinetochore function. As expected, Plk1wt restored chromosome alignment . With Plk1aa or Ch-Plk1aa, however, chromosome alignment remained impaired. Thus, a functional PBD is required for Plk1 to align mitotic 10 34 chromosomes, consistent with earlier reports,. Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 4 A second loss-of-function phenotype of Plk1 is lagging anaphase chromosomes, which occur at very modest losses of Plk1 activity after chromosome alignment on a bipolar 12 spindle and silencing of the mitotic checkpoint. We tested if delocalized Plk1 was sufficient to restore accurate anaphase chromosome segregation. As expected, Plk1wt localized and restored chromosome segregation compared to vector cells, yet both Plk1aa and Ch-Plk1aa failed to do so . We conclude that PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19856273 PBD-dependent kinase localization is required for both chromosome alignment and segregation. Recapitulating Plk1 at the kinetochore We next considered how Plk1 localization within the kinetochore might control function. At the extremes, Plk1 could bind each substrate directly or it could bind a single p.