![Msp1-mediated phosphorylation of Megator disrupts its interaction with Mad1.(A and B) Western blot analysis of Mad1 immunoprecipitates (IP; A) and Megator hyperphosphorylation (B) from lysates of asynchronous and mitotically enriched (+ colchicine) control and Mps1-depleted S2 cells. When indicated, lysates were treated with λ-phosphatase (λPP). (C) In vitro kinase assay with recombinant MBP-Megator fragments and GST-Mps1 in the presence of [γ-32P]ATP. (D) Schematic representation of Drosophila Megator (ELM resource) and Clustal Omega (EMBL-EBI) local sequence alignment for the indicated Megator/Tpr orthologues. Residues conservation: *, fully conserved;:, strongly similar properties;., weakly similar properties. Residues phosphorylated by Mps1 (P) were identified by MS analysis after in vitro kinase assay. (E and F) Pull-downs of recombinant MBP-Megator1,187–1,655 fragments by bead-immobilized 6xHis-Mad11–493 or 6xHis-BubR11–358 (negative control; E) and corresponding quantifications (F) from two independent experiments. B, beads; FT, flow-through. (G and H) Immunofluorescence images (G) and schematic representation (H) of EGFP-Megator1,187–1,655 clustering by LARIAT in mitotic S2 cells. EGFP-aPKC was used as negative control. (I and J) Quantification of Mad1 levels at EGFP-Megator1,187–1,655 clusters (n ≥ 114 clusters; I) and at KTs (n ≥ 64 KTs; J). In F, I, and J, data are presented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). Scale bars, 5 µm (inset, 1 µm).](https://cdn.rupress.org/rup/content_public/journal/jcb/219/3/10.1083_jcb.201906039/7/jcb_201906039_fig2.png?Expires=1771362405&Signature=z-w5cpcbMeMFCf5otTP4kgB9XEB6Lo~9yabGpioTl4iQGPQ7M5AX4DhCMNvc68nP9c0u8H~TpsfuZEk0g1NHVLU~8jCyBim8fcBcU~NyShaq0DUJ3das6~GShJjw8rWQiyQbRZd40OuDb6T9NXau~d0ibx6RqWlx8Dx7tPFXeuUVSnkQgCHiNayPHmCXQrr~YEKSMGk4yFOt-vpsHjLqrsuFJOEb-wJPDuiP9Z29uJe0VXWKah9NXiFIl3GhY30Hk0RB3ZZZxSM0UIJ7ZdoOcKQRFlgjtqE9ZCKz6AObcozeKZw~Mdl8awtu2O0GlW~stI8dCr4k3Y8t8X-kINWp~g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Msp1-mediated phosphorylation of Megator disrupts its interaction with Mad1. (A and B) Western blot analysis of Mad1 immunoprecipitates (IP; A) and Megator hyperphosphorylation (B) from lysates of asynchronous and mitotically enriched (+ colchicine) control and Mps1-depleted S2 cells. When indicated, lysates were treated with λ-phosphatase (λPP). (C) In vitro kinase assay with recombinant MBP-Megator fragments and GST-Mps1 in the presence of [γ-32P]ATP. (D) Schematic representation of Drosophila Megator (ELM resource) and Clustal Omega (EMBL-EBI) local sequence alignment for the indicated Megator/Tpr orthologues. Residues conservation: *, fully conserved;:, strongly similar properties;., weakly similar properties. Residues phosphorylated by Mps1 (P) were identified by MS analysis after in vitro kinase assay. (E and F) Pull-downs of recombinant MBP-Megator1,187–1,655 fragments by bead-immobilized 6xHis-Mad11–493 or 6xHis-BubR11–358 (negative control; E) and corresponding quantifications (F) from two independent experiments. B, beads; FT, flow-through. (G and H) Immunofluorescence images (G) and schematic representation (H) of EGFP-Megator1,187–1,655 clustering by LARIAT in mitotic S2 cells. EGFP-aPKC was used as negative control. (I and J) Quantification of Mad1 levels at EGFP-Megator1,187–1,655 clusters (n ≥ 114 clusters; I) and at KTs (n ≥ 64 KTs; J). In F, I, and J, data are presented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). Scale bars, 5 µm (inset, 1 µm).
