Fig. 5: CHK2 Y156 phosphorylation promotes Mps1 T288 phosphorylation and kinetochore localization.

A Interaction of CHK2 Y156F with Mps1 was reduced as compared to that observed with CHK2 WT and CHK2 Y156E. Co-transfection and co-IP were performed with HEK293T cells. Cells were treated with 50 ng/ml nocodazole overnight before collection. B Nocodazole-induced Mps1 T288 phosphorylation was decreased in CHK2 Y156F HeLa cells. Cells were treated with nocodazole overnight. C, D Mps1 stability was lower with Y156F CHK2 than with WT and Y156E CHK2 coexpression in HEK293T cells. Cells were treated with 50 μg/ml cycloheximide (CHX) and collected at the indicated time points (C). Mps1 bands were quantified and normalized to those of Actin. Mean ± SD from four independent experiments is shown in (D). E, F Kinetochore localization of Mps1 was impaired in CHK2 Y156F HeLa cells. CHK2 WT or Y156F HeLa cells were treated with nocodazole overnight and processed for confocal microscopy by co-staining with anti-Mps1 and anti-CENPB (a kinetochore marker) antibodies. Representative images are shown in (E). Scale bar, 5 μM. Percent cells with more than five colocalized kinetochores were determined and are shown in (F). G Interaction between HEC1 and Mps1 was comparatively higher in the stable cell line with WT CHK2 (#103) than that observed in Y156F CHK2 (#A2). Cells were treated with nocodazole overnight.