Fig. 2: DYRK2 phosphorylates USP28 but promotes its degradation through a kinase activity-independent mechanism.

A HEK-293T cells were transfected with a gradient of wild-type (WT) or kinase-dead version (KM) of Flag-DYRK2 and USP28 protein levels were determined by WB. B HeLa cells were transfected or not with Flag-DYRK2 WT and treated or not with the DYRK2 inhibitor LDN192960 (5 μM) for 2 h. C HeLa cells were transfected or not with a gatekeeper version of DYRK2 (GK) and treated or not with PP1 Analog (3 μM) for 3 h. In A, B and C, protein levels were analyzed by WB, and HSF1 phosphorylation at S320 was used as a control for DYRK2 activity. D MDA-MB-468 DYRK2-knockout cells were transfected with the indicated plasmids and treated with MG-132 (10 μM) for 12 h. USP28 Ser/Thr phosphorylation levels were analyzed by WB on the Flag-USP28 immunoprecipitates. E USP28 human recombinant protein was incubated in presence or absence of DYRK2 human recombinant protein, ATP and λ-phosphatase. Electrophoretic mobility was determined by WB. Changes in DYRK2 mobility are due to auto-phosphorylation. F 2D and 3D scheme of USP28 protein indicating the different motifs and domains of the protein in color code (see legend) and in yellow the phospho-residues identified by MS/MS in the presence of DYRK2. G HEK-293T cells were transfected with the indicated plasmids and analyzed by WB. Note: a significant result is shown of at least 3 biological replicates.