Fig. 5

Patient-derived PDK1 mutants block CK1/GSK3-mediated PDK1 phosphorylation and SPOP-mediated PDK1 degradation. A A schematic presentation of the patients associated PDK1 mutations occurred around GSK3 mediated PDK1 phosphorylation region. B HEK293T cells transfected with indicated constructs were resolved by phospho-tag SDS-PAGE and immunoblotted with indicated antibodies. C IB analysis of WCL derived from HEK293T cells transfected with Flag-PDK1 (WT, S390L, S394L, S398L) and with/without HA-GSK3β-S9A. The relative protein levels of PDK1 were normalized with GAPDH. D IB analysis of WCL and GST-pulldown products derived from HEK293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 10 h before harvested. E IB analysis of WCL derived from HEK293T cells transfected with Flag-PDK1 (WT, S390L, S394L, S398L) and with/without HA-SPOP. The relative protein levels of PDK1 were normalized with GAPDH. F–H IB analysis of DLD1-PDK1 knockout cells stably infected with indicated constructs. Resulting cells were subjected to colony formation and soft agar assays (G), and the relative colony numbers were quantified (H). (mean ± SD, n = 3) (t test), *P < 0.05, **P < 0.01. I-K Cells generated in (F) were subjected to mouse xenograft assays. Tumor sizes were monitored (I), and dissected tumors were weighed (J, K). (mean ± SD, n = 8) (for I, and t test for K), *P < 0.05., **P < 0.01. L IB analysis of WCL derived from dissected tumor tissues