Cdc5 phosphorylates Stu2 ^T866 . (A) Schematic illustrating activation of kinases at the kinetochore by chemical-genetic tethering. Ndc80 and Spc24 are shown in light grey, and Nuf2 and Spc25 are shown in dark grey. In the presence of rapamycin, the kinase-FRB fusion is induced to bind Nuf2-FKBP12, localizing the kinase to the kinetochore. If the kinase is able to phosphorylate Stu2^T866, we postulated that Stu2 will unbind Ndc80c and delocalize from the kinetochore. (B) Exponentially growing stu2-AID cells harboring TOR1-1 fpr1Δ NUF2-FKBP12 CDC5-FRB and an ectopic STU2-GFP variant (STU2^WT-GFP, M4968; stu2^T866V-GFP, M4969) were arrested in α-factor for 3 h. Cells received either 500 µM auxin + 200 ng/ml rapamycin or 500 µM auxin + DMSO for 30 min prior to being fixed and imaged. Stu2-GFP puncta intensity was quantified using ImageJ. Bars are an average of n = 114–166 individual measurements. Error bars are SEM. P values from an unpaired t test. (C) Exponentially growing cells harboring endogenous STU2-GFP and pMET-CDC20, with or without the cdc5-1 temperature-sensitive allele (CDC5^WT, M2827; cdc5-1, M3138), were cultured in methionine-containing media to arrest cells in metaphase for 2 h. Cells were then transferred to 37°C to inhibit cdc5-1 for 1 h. Cells were fixed and imaged to determine kinetochore-proximal Stu2-GFP. Bars are an average of n = 106–116 individual measurements. Error bars are SEM. P values from two-tailed unpaired t tests. (D) Cdc5:Stu2-binding assay. Anti-FLAG magnetic beads were incubated with control (untagged, M3) or Cdc5-FLAG (M4032) lysate and subjected to anti-FLAG IP. Purified Stu2-V5 protein was incubated with beads for 30 min, washed, and eluted with SDS-PAGE sample buffer. Samples were analyzed by SDS-PAGE and immunoblotting. (E) Phosphorylated serine and threonine residues identified by mass spectrometry from Stu2-V5 IP. Exponentially growing cells harboring TOR1-1 fpr1Δ NUF2-FKBP12 CDC5-FRB stu2-AID and STU2^WT-V5 (M5003) were treated with either 500 μM auxin + DMSO or 500 μM auxin + 0.05 μg/ml rapamycin 30 min prior to harvesting, lysed to produce protein sample, subjected to α-V5 IP, and analyzed by mass spectrometry. Table shows which phospho-residues were identified in each sample, as well as the putative kinase and reference for each phosphorylation event. (F) Cells harboring TOR1-1 & fpr1Δ (M1375) and TOR1-1 fpr1Δ NUF2-FKBP12 CDC5-FRB stu2-AID cells with either STU2^WT-GFP (M4968) or stu2^T866V-GFP (M4969) were serially diluted and spotted on plates containing DMSO, 500 μM auxin, 0.05 μg/ml rapamycin, or 500 μM auxin + 0.05 μg/ml rapamycin. Note some panels appear in Fig. S3 H. IP, immunoprecipitation. Source data are available for this figure: SourceData F3.