Figure 2.

Phosphoacceptor Site Mutants Display Minimal Changes in the Pattern of RET AutoP

(A) WB analyses of recombinant purified RET ICD (2.5 μM) WT and the indicated Y to F mutants treated with ATP (5 mM) and MgCl₂ (10 mM) for 0–120 min using the indicated antibodies.

(B) Enzymatic assay performed with RET ICD WT, Y900F, Y905F, or the double mutant Y900/905F incubated with increasing concentrations of the RET Y1015 KRRDYLDLAS peptide. Data shown represent the mean (OD/min) ± SE, n = 4.

(C) Catalytic efficiency constants (k_cat/K_M) of (B).

(D–G) Dissection of Y1015 and Y1029 autoP by LFQMS analysis of recombinant purified RET ICD WT and Y1015F or Y1029F mutants (2.5 μM) treated with ATP (5 mM) and MgCl₂ (10 mM). Data represent the mean signal value (Log₂ ratios of phosphorylated peptides standardized to the nonphosphorylated counterparts) ± SE, n = 4.

See also Figure S2 and Table S1.