Figure 4

Activity of purified Ack1 kinase domain. A, purified Ack1 kinase domain is active in vitro. The catalytic activity of Ack1 kinase domain was measured towards a WASP-derived peptide at different enzyme concentrations. The figure is representative of three experiments. B, increasing local concentrations of purified Ack1 kinase domain at the surface of lipid vesicles stimulates autophosphorylation. The purified dephosphorylated Ack1 kinase domain was incubated in the presence or absence of ATP and in the presence of lipid vesicles containing different mole ratios of DOGS NTA-Ni. After the reaction, equal amounts of Ack1 kinase domain were separated by SDS-PAGE and analyzed by Western blotting using anti- phospho-Ack1 (pY284) and anti His₆ antibodies. In these experiments, the enzyme concentration was 142 nM and the Ni-NTA was kept constant at 58.5 μM. In order to keep the Ni-NTA concentration constant, the amount of total lipids was varied inversely with the mole % of Ni-NTA in the vesicles. For 0% and 2% Ni-NTA, the total lipid concentration was 3.3 mM and for 5% Ni-NTA the lipid concentration was 1.3 mM. No ATP/No ves: reaction in the absence of vesicles and ATP. No ves: reaction in the absence of vesicles. The figure is representative of three experiments. C, quantitation of the western blots described in B. Densitometry readings were used to calculate the ratios of phosphorylated Ack1 (pY284 blot) to Ack1 (His₆ blot). Error bars indicate standard error.