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Figure 5


Fig. 5. Analysis of a total MARK3 phosphorylation-site mutant. (A,B) Wild-type (wt) and indicated mutant forms of GST-MARK3 (ki, kinase inactive D196A mutant), were expressed in HEK 293 cells, purified on glutathione-Sepharose and analysed by western blot with regards to total MARK3 protein ({alpha}GST), T-loop phosphorylation ({alpha}p-T211) and 14-3-3 binding ({alpha}14-3-3). In the 17A-MARK3 mutant, the 17 phosphorylation sites, indicated in Fig. 3 (S42, S45, T61, T211, S378, S396, S400, S419, S469, T491, T507, T536, T549, T564, T576, S583, S619), were converted to alanine residues. 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 17A+T211-MARK3 mutant is identical to the 17A mutant, except that it has an intact T211 in the T-loop. In vitro kinase activity was determined using the protein substrate Cdc25c or the AMARA peptide substrate. The results are presented as the mean of a triplicate (AMARA) or duplicate (Cdc25c) sample + s.d., and are representative of at least three experiments.





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