Fig. 2. Biochemical characteristics of EhPAK. (A) Interaction of truncated EhPAK with small GTPases Rac1 or Cdc42 GST-hybrid proteins carrying —C-PAK (5 µg), —N-PAK (5 µg), —Np-PAK (5 µg) and —WASP (0.5 µg) coupled to glutathione Sepharose beads were incubated with purified recombinant GTPases (1.5 µg), human Rac1 or human Cdc42, activated (+, GTPS loaded) or not (—, loaded with GDP). The potential complex (hybrid protein)/GTPase was separated by electrophoresis, transferred to nitrocellulose, and the presence of GTPases was revealed by immunoblotting with mAbRac1 or mAbCdc42 antibodies. (B) Kinase activity of the C-terminal domain of EhPAK. The hybrid protein GST-C-PAK (1 µg) immobilised on glutathione Sepharose beads and thrombin-cleaved derivative soluble protein C-PAK (500 ng) and uncoated glutathione Sepharose beads were subjected to an in vitro kinase assay using myelin basic protein (MBP) as the substrate in the presence of [-³²P]ATP. Samples were run on a gel and analysed with a phosphoimager, and the phosphorylation was quantified in arbitrary units with the IQ Mac V12 molecular imaging system. An autophosphorylation background of MBP is shown in the control line and a large increase in MBP phosphorylated was obtained when the carboxylic domain of EhPAK was added.

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