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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 ${gamma}$ in the absence or presence of microcystin-LR. The GST-MARK3, which also carried an HA-tag, was subsequently immunoprecipitated using ${alpha}$ HA antibodies, washed and analysed by western blot with regards to total protein ( ${alpha}$ GST), T-loop phosphorylation ( ${alpha}$ p-T211) and 14-3-3 binding ( ${alpha}$ 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 ( ${alpha}$ GST) and 14-3-3 binding ( ${alpha}$ 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 ${zeta}$ 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 ${zeta}$ , were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blotting with regards to total protein ( ${alpha}$ GST), and binding to endogenous MARK3 ( ${alpha}$ MARK3), Raf ( ${alpha}$ Raf) and QSK ( ${alpha}$ 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).

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