PLK1 phosphorylates MLL5 on Ser-861. (A) Gel mobility shift assay of MLL5-CD and MLL5-CD4. 293T cells expressing truncated FLAG-MLL5 mutant were treated with RO3306 or nocodazole in the presence or the absence of BI2536. The cell lysates were detected by Western blotting (WB) with anti-FLAG antibody. (B) PLK1 phosphorylation of MLL5 in vitro. (top) 32P autoradiograph of in vitro PLK1 kinase assay. GST-MLL5-CD-4 purified from E. coli was subjected to an in vitro PLK1 kinase assay. BI2536 was introduced as an acute inhibitor of PLK1. GST and casein served as negative and positive controls, respectively. (bottom) Coomassie brilliant blue staining for GST, GST-MLL5-CD4, and Casein. (C) Gel mobility shift assay of MLL5-CD4-S861A and MLL5-CD4-TST887AAA. 293T cells expressing FLAG-MLL5-CD4 mutant were treated with RO3306 or nocodazole in the presence or absence of BI2536. Cell lysates were detected by WB with anti-FLAG antibody. Arrows in A and C indicate the slowest-migrating form of FLAG-MLL5 mutant. Arrowheads in A and C indicate the fastest-migrating form. Diagram in A and C illustrates the respective MLL5 migration patterns for the blot on the left. Results are representative of at least three experimental repeats.
