FIGURE 1.

The canonical Wnt pathway kinase CK1α interacts with and phosphorylates Jade-1S. A, HEK 293T cells were transiently transfected with the indicated plasmids for 24 h. V5.Jade-1 coprecipitated with the FLAG-tagged Wnt-pathway kinases F.GSK3β and F.CK1α as well as a positive control protein (F.NPHP4) but not a negative control protein (F.EPS^1–225). Only coexpression of F.CK1α generated a larger band indicative of multiple posttranslational modifications of V5.Jade-1. IB, immunoblot. B, endogenous CK1α or a control protein (Densin) was immunoprecipitated from confluent HEK 293T cells. Both the long and short isoforms of endogenous Jade-1 coprecipitated with CK1α but not Densin despite equal protein amounts in the IP lysate. Asterisk, Jade-1; h.c., heavy chain. C, recombinant purified GST.CK1α was incubated with or without a bacterially expressed and purified recombinant truncation of His.Jade-1 (4–174). Reactions were diluted with IP buffer and incubated overnight with Ni²⁺ beads. GST.CK1α coprecipitated only with Ni²⁺ beads plus His.Jade-1 4–174. D, plasmids were transiently transfected as indicated in HEK 293T cells for 24 h prior to harvesting cells as a whole cell lysate (WCL). Coexpression of F.CK1α stabilized protein expression of V5.Jade-1 but increased band size in contrast to the reduced band size observed when coexpressed with F.NPHP4. E, whole cell lysates of HEK 293T cells transiently transfected for 24 h with the indicated plasmids prior to 18-h exposure to kinase inhibitor or dimethyl sulfoxide (DMSO) control. The CK1-specific kinase inhibitor D4476 reduces band size of V5.Jade-1 in contrast to the use of dimethyl sulfoxide alone or a control kinase inhibitor, BI 2536. F, plasmids were transiently transfected as indicated in HEK 293T cells and processed 24 h later as a whole cell lysate. Coexpression of a kinase-dead mutant of F.CK1α (K46D) failed to stabilize the larger expression form of V5.Jade-1.