Fig. 5. MLK3 phosphorylates the N-terminal head region of golgin-160. HeLa cells were transfected with GFP golgin-160 mutants encompassing specific amino acid regions of the protein. The golgin-160 proteins were immunoprecipitated and incubated with expressed MLK3 isolated from CHO cells and phosphate incorporation was determined in a kinase assay. (A) (Top) GFP immunoblot of golgin-160 proteins in the kinase reaction mixture after incubation for 60 minutes in the absence or presence of MLK3. (Bottom) Relative phosphate incorporation into golgin-160 wild-type and mutant proteins in the absence (open bars) or presence (closed bars) of MLK3. The phosphorylation of the golgin-160 mutants was normalized to the phosphorylation of golgin-160 wild type in the absence of MLK3, which was set at a value of 1. (B) (Top) Immunoblot of GFP/golgin-160(1-393) and GFP/golgin-160(393-1498) in the kinase reaction mixture after incubation in the absence or presence of MLK3 for 20 minutes or 40 minutes. (Bottom) Phosphate incorporation in the golgin-160(1-393) mutant incubated in the absence (open bars) or presence (closed bars) of MLK3 for 20 or 40 minutes. No phosphate incorporation was observed in the golgin-160(393-1498) fragment, even though the expression levels for both mutant proteins were the same (top). MLK3 increased phosphorylation of golgin-160 wild type and the 1-393 N-terminal mutant by 2.5-6.2 times in three or four separate experiments.