1. PDK1 regulates PLK1 in vivo and in vitro.
PDK1 regulates PLK1 in vivo and in vitro. A, immunoblot analysis of indicated proteins in HCT116 PDK1 wild-type (PDK1+/+) and knockout (PDK1−/−) cells. Cells were synchronized by double-thymidine block and released into cell cycle at indicated times. B, immunoblot analysis of indicated proteins in MDA-MB-231 shNC and PDK1 knockdown (shPDK1) cells. Cells were synchronized by double-thymidine block and released into cell cycle at indicated times. C, cells were synchronously released from double-thymidine arrest (TT) and harvested at the indicated times for FACS analysis. Percentages of cells positive for phosphor-H3 (S28) are indicated. PI, propidium iodide. D, PLK1 protein domain analysis (top) and PDK1 consensus motif alignment with other known PDK1 substrates (bottom). E, immunoblot analysis of immunoprecipitated PLK1 in 293T cells transfected with PLK1, or cotransfected with PDK1, with or without 2.5 μmol/L BX795, 5.0 μmol/L BX912, and 1.0 μmol/L VX680 treatment for 24 hours. F, in vitro immunoprecipitation-kinase assay using recombinant PDK1 and immunoprecipitated endogenous PLK1 from DLD1 cells as substrate. Cells were synchronized by a double-thymidine arrest and released in the presence or absence of 2.5 μmol/L BX795 or 1.0 μmol/L VX680 for 8 hours. PLK1 IP-kinase assay was conducted and the phosphorylation of PLK1 was assessed by using p-T210 PLK1 antibody. G, in vitro kinase assay using recombinant PDK1 and recombinant PLK1 with or without 1.0 μmol/L BX795, 1.0 μmol/L BX912, and 1.0 μmol/L VX680.
Jing Tan et al. Cancer Discovery 2013;3:
©2013 by American Association for Cancer Research