Fig. 6. The STRAD pseudokinase is capable of binding ATP. (A) The wild-type and mutant GST-STRAD proteins were incubated with increasing concentrations of [-³²P]ATP, in the presence or absence of 5 mM Mg²⁺, and binding of ATP to the proteins was measured as described in the Materials and Methods. Results shown are the means of three separate experiments carried out in duplicate. Data were fitted to a single-site binding model: bound=[ATP]/(K_d+[ATP]). (B,C) Displacement of ATP from wild-type STRAD by ADP (B) or AMP (C). A fixed concentration of [-³²P]ATP (200 µM) was incubated with the GST-STRAD protein in the presence of increasing concentrations of either ADP or ATP, and in the presence or absence of 5 mM Mg²⁺; binding of ATP to the proteins was measured as described in the Materials and Methods. Data were fitted to the binding models: bound=[ATP]/([ATP]+ K_{d ATP}(1+[ADP]/K_{d ADP})) or bound=[ATP]/([ATP]+K_{d ATP}(1+[AMP]/K_{d AMP})). (D) HEK293 cells were transfected with the indicated constructs and analysis performed as described in the legend to Fig. 1A. Results shown are the mean±s.d. of two independent assays carried out in triplicate. (E) HeLa cells were transfected with the construct encoding wild-type or indicated mutant of Flag-STRAD in the presence of GFP-LKB1 and Myc-MO25, and analysed as described in the legend to Fig. 3.