Figure 2. ACK1 directly phosphorylates cortactin at Y421/466/482.

(A) Characterization of purified ACK1. Recombinant 6X-histidine tagged recombinant ACK1 was purified from SF9 cells and 5 micrograms stained with either Coomassie blue (left) or analyzed by Western blotting with anti-ACK antibodies (middle). Kinase activity was assayed by analyzing autophosphorylation (Autophos) using 0.5 micrograms of 6X-His ACK1 incubated with gamma-³²P-ATP for 10 min at 30°C. The reaction was evaluated by SDS-PAGE and autoradiography (right). The position of molecular weight standards is shown on the left and 6X-His ACK1 on the right. (B) Identification of cortactin tyrosine residues phosphorylated by ACK1. Purified GST-cortactin murine wild type (WT) or Y421/466/482F (triple tyrosine mutant; TYM) (0.5 micrograms) were incubated in kinase assays without or with 3U of purified Src (left) or with the indicated amounts of purified 6X-His ACK1 (middle and right) in the presence of [gamma-³²P]ATP. Reactions were separated by SDS-PAGE and analyzed by audioradiography. Arrows indicate the positions of phosphorylated GST-cortactin and 6X-His ACK1 (left). (C) Determination of the K_M of ACK1 for cortactin. Graphical representation showing ACK1 phosphorylation kinetics for cortactin calculated from the kinase assay in B (right panel). Percent of maximum phosphorylation signal measured by densitometry is represented on the ordinate versus concentration of ACK1 (in micrograms) on the abscissa. The calculated K_M for cortactin phosphorylation is shown. Data in B and C are representative from three independent experiments. (D) ACK1 knockdown reduces cortactin phosphorylation on tyrosine 421. 1483 cells transfected with scrambled control (Ctl) siRNA or ACK1-targeting siRNA were lysed and analyzed by Western blotting for ACK1 knockdown (ACK1) and cortactin pY421 phosphorylation (Cort pY421). Beta actin was blotted to verify equivalent protein loading. Blots are representative of two independent experiments.