Fig. 4. Analysis of single MARK3 phosphorylation-site mutants. (A,B) Wild-type (wt) and indicated phosphorylation-site-mutant forms of GST-MARK3, were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total MARK3 protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3). In vitro kinase activity was determined using the AMARA peptide substrate. S619 was not identified in our phosphopeptide mapping analysis but was mutated, because the equivalent site on xPAR-1 was reported to be phosphorylated by aPKC (Kusakabe and Nishida, 2004). The results are presented as the mean of a duplicate sample +s.d., and are representative of two experiments.