Inhibition
and reactivation of the steady-state activity of SHP2
variants. (A) Domain cartoon of the SHP2cat construct.
(B) Domain cartoon of the SHP21–526 construct (SH2N, cyan; SH2C, blue; CAT, orange). (C, D) The steady-state
activity of SHP2 variants (C, SHP2237-529; D, SHP21–526; WT, black squares, N308D variant, red diamonds)
were measured after incubating the samples with the indicated concentrations
of H2O2 for 15 min. The % remaining activity
was calculated by normalizing the measured activity to the sample
incubated without H2O2. (E, F) The ability of
oxidized SHP2 variants (E, SHP2237-529; F, SHP21–526) to be subsequently reactivated was determined
by measuring the activities of reduced (no H2O2) and oxidized (15 min incubation with 500 μM H2O2 prior to activity measurements) SHP2 samples in the
presence of the indicated concentrations of DTT. The % reactivation
was calculated by normalizing the activities of the oxidized samples
to the corresponding reduced samples. ±SE, n = 3–4. Two-way ANOVA test, with ****p <
0.0001 (C, D) or *p < 0.05 (F) between WT and
mutant.