Ve delocalized Plk1aa was unable to rescue. We conclude that

Ve delocalized Plk1aa was unable to rescue. We conclude that Kif2c-tethered Plk1 rescues the chromosome alignment function, either due to activity at microtubule tips or at the inner centromere. Next, we tested anaphase chromosome segregation. As before, Plk1C-Kif2c rescued accurate chromosome segregation whereas outer-kinetochore Plk1 failed to do so. However, chromatin-localized Plk1 also functionally restored chromosome segregation. Again, rescue required active kinase, and was reversed with BI-2536. Moreover, soluble Plk1aa was unable to rescue. Tethered constructs did not disrupt chromosome alignment or PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 segregation in the absence of inhibition of Plk1as by 3-MB-PP1. Thus Plk1 acts at either chromatin or at Kif2c sites to elicit its function for proper chromosome segregation in anaphase. Moreover, the functionally distinct behavior of Plk1C-H2B from Plk1C-Kif2c in chromosome alignment suggests that Plk1 phosphorylates one or more unique substrates to execute the disparate functions. Re-evaluating Plk1 function at the outer kinetochore We identified kinetochore functions that arise from Plk1 signals at chromatin or the inner centromere, where the bulk of Plk1 is located. However, outer kinetochore activity could be important if Plk1C-Kif2c 518303-20-3 web operates at the microtubule tips, and Plk1C-Dsn1 might fail to restore activity merely due to impaired access to key outer kinetochore substrates. To address the first question, we separated Plk1C-Kif2c functions at the inner centromere versus outer kinetochore. The Kif2c N-terminus is important for inner centromere targeting40 and the SxIP sequence within the N-terminus promotes microtubule tip37 38 tracking,. Using Kif2c localization mutant constructs, both N-terminal deletion mutant and the microtubule tip-tracking mutant were expressed as Varlitinib chemical information stable Plk1 fusions and exhibited catalytic activity similar to Plk1C-Kif2cwt. As expected, the SKNN mutant, but not the N mutant, localized to the inner centromere and the SKNN mutant failed to localize at the outer kinetochore. Notably, the SKNN, but not the N mutant, rescued both chromosome alignment and anaphase chromosome segregation phenotypes, and rescue depended on tethered Plk1 Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 7 activity, as seen by BI-2536-dependent reversal. Thus, Plk1C-Kif2c operates in the inner centromere to execute key mitotic functions. We next considered that Plk1C-Dsn1, though localized correctly and catalytically active, might not access key outer kinetochore substrates. As a measure of Plk1 activity at the outer kinetochore, we evaluated BubR1 phosphorylation, which is detectable by a slower 18 33 migrating band on SDS-PAGE,. Therefore, we tested BubR1 phosphorylation from mitotic extracts, +/- 3-MB-PP1 and found that, indeed, Plk1C-Dsn1 did not reach or only weakly phosphorylated BubR1. Therefore, we generated additional constructs, tethering Plk1 to the outer kinetochore proteins Bub1, BubR1 and Hec1. We identified stable cell lines exhibiting diverse expression levels including ones expressed at high levels or similar levels to the other tethers with comparable catalytic activity. Moreover, the Plk1C-Bub1 and Plk1CHec1 constructs appeared to restore the slow-mobility p-BubR1 band. Nevertheless, all three constructs failed to rescue either chromosome alignment or segregation. Together, these data support the idea that outer kinetochore Plk1 activity is not required for its function in ch.Ve delocalized Plk1aa was unable to rescue. We conclude that Kif2c-tethered Plk1 rescues the chromosome alignment function, either due to activity at microtubule tips or at the inner centromere. Next, we tested anaphase chromosome segregation. As before, Plk1C-Kif2c rescued accurate chromosome segregation whereas outer-kinetochore Plk1 failed to do so. However, chromatin-localized Plk1 also functionally restored chromosome segregation. Again, rescue required active kinase, and was reversed with BI-2536. Moreover, soluble Plk1aa was unable to rescue. Tethered constructs did not disrupt chromosome alignment or PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 segregation in the absence of inhibition of Plk1as by 3-MB-PP1. Thus Plk1 acts at either chromatin or at Kif2c sites to elicit its function for proper chromosome segregation in anaphase. Moreover, the functionally distinct behavior of Plk1C-H2B from Plk1C-Kif2c in chromosome alignment suggests that Plk1 phosphorylates one or more unique substrates to execute the disparate functions. Re-evaluating Plk1 function at the outer kinetochore We identified kinetochore functions that arise from Plk1 signals at chromatin or the inner centromere, where the bulk of Plk1 is located. However, outer kinetochore activity could be important if Plk1C-Kif2c operates at the microtubule tips, and Plk1C-Dsn1 might fail to restore activity merely due to impaired access to key outer kinetochore substrates. To address the first question, we separated Plk1C-Kif2c functions at the inner centromere versus outer kinetochore. The Kif2c N-terminus is important for inner centromere targeting40 and the SxIP sequence within the N-terminus promotes microtubule tip37 38 tracking,. Using Kif2c localization mutant constructs, both N-terminal deletion mutant and the microtubule tip-tracking mutant were expressed as stable Plk1 fusions and exhibited catalytic activity similar to Plk1C-Kif2cwt. As expected, the SKNN mutant, but not the N mutant, localized to the inner centromere and the SKNN mutant failed to localize at the outer kinetochore. Notably, the SKNN, but not the N mutant, rescued both chromosome alignment and anaphase chromosome segregation phenotypes, and rescue depended on tethered Plk1 Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 7 activity, as seen by BI-2536-dependent reversal. Thus, Plk1C-Kif2c operates in the inner centromere to execute key mitotic functions. We next considered that Plk1C-Dsn1, though localized correctly and catalytically active, might not access key outer kinetochore substrates. As a measure of Plk1 activity at the outer kinetochore, we evaluated BubR1 phosphorylation, which is detectable by a slower 18 33 migrating band on SDS-PAGE,. Therefore, we tested BubR1 phosphorylation from mitotic extracts, +/- 3-MB-PP1 and found that, indeed, Plk1C-Dsn1 did not reach or only weakly phosphorylated BubR1. Therefore, we generated additional constructs, tethering Plk1 to the outer kinetochore proteins Bub1, BubR1 and Hec1. We identified stable cell lines exhibiting diverse expression levels including ones expressed at high levels or similar levels to the other tethers with comparable catalytic activity. Moreover, the Plk1C-Bub1 and Plk1CHec1 constructs appeared to restore the slow-mobility p-BubR1 band. Nevertheless, all three constructs failed to rescue either chromosome alignment or segregation. Together, these data support the idea that outer kinetochore Plk1 activity is not required for its function in ch.