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Figure 1


Fig. 1. (A) Identification of GST-MARK3 binding partners. Wild-type GST-MARK3 was expressed in HEK 293 cells, purified on glutathione-Sepharose, and subjected to SDS-PAGE. The gel was stained with colloidal Coomassie Blue, and protein bands were excised, washed and digested with trypsin. The identity of the interacting proteins was determined by mass spectrometry, as described previously (Al-Hakim et al., 2005). eIF1{gamma} (eukaryotic elongation factor 1{gamma}) is a non-specific binding-protein, observed to interact with most GST-fusion proteins expressed in HEK293 cells. (B) Analysis of various MARK mutants with regards to 14-3-3 binding and in vitro kinase activity. Wild-type (wt) or indicated mutant forms of GST-MARK3 (ki, kinase inactive D196A mutant; kd+UBA, kinase domain with ubiquitin-associated domain, residues 1-382), were expressed in HEK 293 cells, purified on glutathione-Sepharose 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). In vitro kinase activity was determined using the Cdc25c protein or AMARA peptide as substrate. The results are presented as the mean of a triplicate sample +s.d., and are representative of at least three experiments.