Fig. 5: Aurora A and B kinases activate CENP-E by releasing it from an auto-inhibited state.
From: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly
a Schematic representation of the CENP-E constructs used in this study. b Co-immunoprecipitation of FLAG-GFP-CENP-E (1779-2701) construct with WT and T422A CENP-E-MYC (1-859). Four independent experiments showed similar results. c Representative maximum intensity-projected point-scanning confocal images of monopoles in U2OS CENP-E WT and T422A cell lines following the indicated treatments. Scale bar: 10 μm. d Quantification of GFP spindle pole/ total intensity ratio following the indicated treatments in CENP-E WT/T422A monopoles. Values are plotted with mean ± SD. N (number of cells, number of independent experiments) - GFP-CENP-E WT: siCTRL + DMSO (64, 3), siCTRL + GSK923295 (58, 3), siCTRL + Cmpd-A (59, 3), siSPDLY + DMSO (60, 3), siSPDLY + GSK923295 (59, 3), siSPDLY + Cmpd-A (61, 3); GFP-CENP-E T422A: siCTRL + DMSO (60, 3), siCTRL + GSK923295 (60, 3), siCTRL + Cmpd-A (61, 3), siSPDLY + DMSO (60, 3), siSPDLY + GSK923295 (60, 3), siSPDLY + Cmpd-A (58, 3). Note that in the absence of KT-bound dynein (CENP-E-WT + siSpindly) chromosomes are dispersed away from the pole by polar ejection forces (PEFs)16, lowering the ratio of polar and total intensity. Statistical significance was determined by the Mann–Whitney U-test (unpaired, two-tailed; no normal distribution). p values are indicated. e Spinning-disk confocal time-series of mitosis of GFP-CENP-E WT/T422A cells undergoing indicated treatments. Red arrowheads show spindle pole accumulation of non-phosphorylated CENP-E and inhibited CENP-E. Time: hour:min. Scale bar: 10 μm. f Illustrated model of the effects of CENP-E phosphorylation by Aurora A and B kinases. CENP-E activation is regulated by phosphorylation/dephosphorylation. Phosphorylation-activated CENP-E is dominant over dynein and thus facilitates chromosome congression to the spindle equator. CENP-E dephosphorylation inactivates CENP-E, thereby triggering dynein-mediated stripping of corona proteins toward the spindle pole. Re-activation of CENP-E by Aurora A phosphorylation at centrosome releases corona proteins from the spindle pole. Illustrations created with BioRender.com.
