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. Author manuscript; available in PMC: 2021 Feb 10.
Published in final edited form as: Cancer Cell. 2020 Feb 10;37(2):200–215.e5. doi: 10.1016/j.ccell.2020.01.001

Figure 3. AURKB Enhances MYC Protein Stability through the S67 Phosphorylation.

Figure 3.

(A) Time-course analysis of MYC p-T58 by immunoblot upon AZD1152 (50 nM) treatment in CUTLL1 and HPB-ALL cells, with ACTIN as a loading control.

(B) Protein interaction between GSK3β and MYC in the presence of AURKB. pCDH-Flag-GSK3β (1 μg), pCDH-HA-MYC (1 μg), and/or increasing doses of pCDH-AURKB (0.5,1, and 2 μg) were transfected into 293T cells for 48 h as indicated. Cell lysates were then subjected to co-IP and immunoblot. The star (*) designates immunoglobulin G heavy chain.

(C) Time-course analysis of HA-MYC levels in 293T cells expressing ectopic HA-MYC, Flag-AURKB WT, or mutant (K106R) as indicated. MYC proteins were quantified and plotted on the right. Data shown are means (±SD) of three independent experiments.

(D) Peptide sequence alignment of MYC (amino acids 58–74) in various species. The S67 site resides in the AURKB phosphorylation consensus motif.

(E) Time-course analysisofHA-tagged MYC(WT ormutants) byimmunoblot in 293T cells(left). MYC proteinswerequantified and plotted ontheright. Datashown are means (±SD) of three independent experiments.

(F) Co-IP of Flag-GSK3β and HA-MYC (WT, S67D, or S67A) from lysates of 293T cells overexpressing respective tagged proteins.

(G) In vitro kinase analysis of recombinant GST-MYC. Active human AURKB proteins were incubated with GST-MYC for kinase reaction. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. Loading controls were shown as Coomassie blue staining in the bottom panels.

(H) Detection of endogenous MYC p-S67. CUTLL1 and primary T-ALL cells were treated with Nocodazole(Noco, 1 μg/mL) for 4 h or AZD1152 (10 nM) for 24 h as indicated. Noco-treated cell lysates were treated with calf-intestinal alkaline phosphatase (CIP) for 30 min before immunoblot. Histone 3 p-S10 (p-S10 H3) levels were shown as a positive control reflecting AURKB activity.

See also Figure S2.