FIGURE 3.

Lys¹⁴⁰ and Lys¹⁴³ are primary MKK4 ubiquitination sites. A: upper panel, locations of lysines within a 100-amino acid region of the MKK4 kinase domain required for ubiquitination. These eight lysines were individually mutated to arginines. Lower panel, immunoprecipitation (IP)/Western blotting of 293T cells transfected with the designated mutants and treated with sorbitol to detect ubiquitinated (Ub; upper) and total (lower) MKK4. B: immunoprecipitation/Western blotting (WB) of 293T cells transfected with wild-type (WT) MAP2K4 or the double mutant K140R/K143R to detect ubiquitinated (upper panel) and phosphorylated (p) and total proteins (lower panels). C: Western blot of CHO cells transfected with wild-type MAP2K4 or the double mutant K140R/K143R. Actin was used as a loading control. Densitometric values are located under the bands and are expressed relative to those of untreated cells, which were set at 1.0.