Fig. 4: ATM and ATR kinases phosphorylate KIFC1-S26 during DNA-damage conditions.
a, b MDA-MB-231 cells were treated with IR or indicated agents for 15 h. The protein and mRNA levels of endogenous KIFC1 and β-actin (as the internal standard) were examined by western blotting (a) and reverse transcription–PCR (b). The data are representative of two independent experiments. a Cycloheximide (CHX, 50 μm mL−1) was added in cells for the indicated time prior to lysis. Relative KIFC1 band intensities were quantified using densitometry and presented. (c) 293 T cells transfected with His-ubiquitin and Flag-KIFC1 plasmids were treated with etoposide (20 μM) or cisplatin (20 μM) for 6 h. MG132 (25 μM) was added for 3 h prior to lysis. Ubiquitinated proteins were precipitated using Ni-NTA beads. KIFC1 ubiquitination was detected by western blot using anti-Flag antibody. d, e MDA-MB-231 cell lysates were immunoblotted with antibodies against KIFC1, ATM, ATR, or β-actin (as the internal standard). d The cells were pretreated with AZD1390 (20 nM), VE-822 (5 μM), AZD6738 (25 nM), MK-8776 (5 μM), or C3742 (10 μM) for 1 h, and then treated with etoposide and these inhibitors for another 15 h. e Cells were infected with shN (Control), ATM-shR, or ATR-shR lentivirus for 72 h and then treated with etoposide for another 15 h. f, j MDA-MB-231 cells were treated with etoposide (20 μM) for 2 h (j) or 4 h (f, j). Endogenous KIFC1 was precipitated using anti-KIFC1 antibody or with IgG (Mock IP), and the precipitated proteins were analyzed by western blotting using the antibody against KIFC1, ATM, ATR, p-ATM (S1981), or KIFC1S26p. g 293T cells transfected with Flag-KIFC1 plasmids were treated with or without etoposide (20 μM) for the indicated time, followed by immunoprecipitation with FLAG-M2 beads. The samples were immunoblotted with antibodies against Flag or p-S/TQ (ATM/ATRsub). h Eight phosphorylation sites of KIFC1 were identified by LC-MS/MS analysis using purified KIFC1 from Flag-IP in 293 T cells. i, k Characterization of KIFC1-S26 phosphorylation antibody (KIFC1S26p, produced in our lab). 293T cells were transfected with indicated Flag-KIFC1 plasmids and followed by immunoprecipitation with FLAG-M2 beads. The precipitated proteins were analyzed by western blot KIFC1S26p or Flag antibodies. k 293T cells were pretreated with AZD1390 or AZD6738 for 1 h, and then treated with etoposide and these inhibitors for another 4 h. The western blot images are representative of 2 independent experiments with similar results. Source data are provided as a Source Data file.
