Fig. 4

CK1/GSK3-mediated PDK1 phosphorylation promotes SPOP interaction with and ubiquitination of PDK1. A IB analysis of WCL and GST-pulldown products derived from HEK293T cells transfected with GST-SPOP, Flag-PDK1 and treated with/without λ-phosphatase. B A schematic presentation of the evolutionarily conserved putative GSK phosphorylation motif in PDK1. Red label indicates the potential GSK3 phosphorylation sites. C HEK293T cells transfected with indicated constructs were resolved by phospho-tag SDS-PAGE and immunoblotted with indicated antibodies. D IB analysis of HEK293T-PDK1 knockout cells stably infected with indicated constructs. Where indicated cells were treated with GSK3β inhibitor CHIR-99021 (10 μM) for 10 h before harvested. E IB analysis of WCL and GST-pulldown products derived from HEK293T cells transfected with indicated constructs. Where indicated, cells were treated with GSK3β inhibitor CHIR-99021 (10 μM) for 10 h before harvested. F A schematic presentation of CK1 phosphorylation motif in PDK1 and other well-established SPOP substrates, such as ERG and SRC-3. Red label indicates the potential CK1 phosphorylation site. G-H HEK293T cell transfected with indicated constructs were resolved by phospho-tag SDS-PAGE (G) or normal SDS-PAGE (H), and immunoblotted with indicated antibodies. Where indicated, cells were treated with CK1 inhibitor IC261 (50 μM) or D4476 (20 μM) for 10 h before harvested. I IB analysis of WCL derived from HEK293T cells transfected with Flag-PDK1 and increasing Myc-CK1δ. Where indicated, cells were treated with CKI inhibitor IC261 (50 μM) or D4476 (20 μM) for 10 h before harvested. J IB analysis of WCL from MCF7 cells transfected with indicated lentiviral shRNA. The transfected cells have been selected in puromycin (1 μg/ml) for 72 h to eliminate the uninfected cells before harvested. K HEK293T cells transfected with indicated constructs were resolved by phospho-tag SDS-PAGE and immunoblotted with indicated antibodies. Where indicated, cells were treated with CK1 inhibitor IC261 (50 μM) or D4476 (20 μM) for 10 h before harvested. L In vitro kinase assay was performed with bacterially purified PDK1 as substrate, and recombinant CK1 protein as the kinase source ³²P isotope-ATP was used for autoradiography of phosphorylated PDK1. M IB analysis of WCL derived from HEK293T cells transfected with Flag-PDK1 (WT, Δ6S) and with/without Myc-CK1δ. N–O IB analysis of WCL and IP products derived from HEK293T cells transfected with Myc-CK1δ and indicated constructs. P A schematic model illustrates that CK1 phosphorylating PDK1 could mediate GSK3 phosphorylating PDK1 and in turn promote SPOP recognizing PDK1