Figure 6.

c-Abl SH3 mutants are resistant to inhibition by Lats2. (a) Mutation of c-Abl at W118 impairs binding to Lats2. HEK293 cells were transfected with the constructs indicated, with 0.25, 0.5, and 1 μg of HA-Lats2 plasmids used. Flag-c-Abl constructs were immunoprecipitated and IP and total extracts were analyzed by immunoblotting. Quantification of the amount of HA-Lats2 that co-immunoprecipitated, normalized to the amount of immunoprecipitated c-Abl, is presented. (b,c) c-Abl T197A and W118A mutants are not inhibited by Lats2. HEK293 cells were transfected with plasmids expressing the proteins indicated. (b) Extracts were analyzed by immunoblotting. (c) The ratio of c-Abl autophosphorylation in the presence of Lats2 versus control (black bars), and the ratio of p73 pTyr⁹⁹ in the presence of Lats2 versus control (gray bars) is presented. The level of c-Abl autophosphorylation was normalized to the amount of total c-Abl, and the level of phosphorylated p73 was normalized to total p73. Ratios were calculated from three independent experiments. The dashed line represents a ratio of 1, where phosphorylation of c-Abl and p73 is the same in the presence or absence of Lats2. Ratios less than 1 indicate inhibition of phosphorylation in the presence of Lats2