Fig. 6: CK1δ binds and phosphorylates USP46 at Thr209, Ser351 and Ser354.

a Volcano plots of USP46 associated proteins identified by mass spectrometric analysis. SK-MEL-103 cells stably expressing FLAG-S-USP46 or Empty Vector were generated and treated with MG132 (10 μM) for 10 h. USP46 or Empty Vector immunoprecipitate complexes were subjected to mass spectrometric analysis (n = 3). b SK-MEL-103 cell lysates were subjected to immunoprecipitation with IgG, anti-CK1δ or anti-USP46 antibodies. The immunoprecipitates were blotted with indicated antibodies. c Purified recombinant GST, GST-CK1δ and His-USP46 were incubated in vitro as indicated. The interaction between CK1δ and USP46 was examined. CBS, Coomassie blue staining. d SK-MEL-103 cells stably expressing empty vector or FLAG-CK1δ were generated. FLAG-CK1δ was immunoprecipitated with anti-FLAG affinity gel and incubated with GST or GST-USP46 protein purified from SK-MEL-103 cells stably expressing empty vector (pLVX3-GST) or pLVX3-GST-USP46 WT in an in vitro kinase buffer with the presence of DMSO or CK1δ inhibitor PF670462 (5 μM). The phosphorylation of USP46 was examined by western blotting using Phos-tag containing gel. e CK1δ phosphorylates USP46 at Thr209, Ser351 and Ser354. Cell lysates of SK-MEL-103 cells stably expressing FLAG-CK1δ were immunoprecipitated with anti-FLAG affinity gel and incubated with GST, GST-USP46 WT, the single mutants or the 3A mutants purified from SK-MEL-103 cells stably expressing empty vector (pLVX3-GST), pLVX3-GST-USP46 WT or indicated mutants in an in vitro kinase buffer. The phosphorylation of USP46 was assessed by western blotting using Phos-tag containing gel. f SK-MEL-103 cells stably expressing shUSP46 were transfected with empty vector (pLV5), USP46 WT or the phosphorylation defective mutant 1A and 3A and western blotting was performed with indicated antibodies. g Endogenous USP46-deficient SK-MEL-103 cells were transfected with empty vector (pLV5), HA-USP46, the single mutants, the 3A mutants or indicated plasmids and then treated with MG132 (10 μM) for 10 h. FLAG-NRAS Q61R was immunoprecipitated with anti-FLAG affinity gel and the ubiquitination of FLAG-NRAS Q61R were measured by western blotting. Source data are provided as a Source Data file.