Fig. 2. (A) Phosphatase treatment of MARK3. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose and treated with PP1 in the absence or presence of microcystin-LR. The GST-MARK3, which also carried an HA-tag, was subsequently immunoprecipitated using HA antibodies, washed and analysed by western blot with regards to total protein (GST), T-loop phosphorylation (p-T211) and 14-3-3 binding (14-3-3 or overlay assay with yeast 14-3-3). (B) Peptide elution of 14-3-3 from the GST-MARK3/14-3-3 complex. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose and, while still coupled to the resin, washed with a phospho-(phospho-Raf) or dephospho-peptide (dephospho-Raf) encompassing the S259 14-3-3 binding site in Raf. The GST-MARK3 was subsequently analysed by western blot with regards to total protein (GST) and 14-3-3 binding (14-3-3). In vitro kinase activity was determined using the Cdc25c protein or the AMARA peptide as a substrate. The results are presented as the mean of a triplicate sample + s.d., and are representative of at least three experiments. (C) Binding of various 14-3-3 mutants to endogenous MARK3, Raf and QSK. (Upper left panel) X-ray crystallographic structure of the 14-3-3 dimer in complex with a Raf peptide, as adapted from (Rittinger et al., 1999). The amphipathic cleft is formed by helix 3 (turquoise), helix 5 (green), helix 7 (red) and helix 9 (orange). (Upper right panel) Enlarged view of the amphipathic cleft with mutated residues indicated. (Bottom panel) Wild-type and indicated mutant forms of GST-14-3-3, were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blotting with regards to total protein (GST), and binding to endogenous MARK3 (MARK3), Raf (Raf) and QSK (QSK). Results shown are representative of two separate experiments. The E180K, F196Y and Y211K mutants studied previously are shown in pink (Benton et al., 2002).