Fig. 3

SHP2 dephosphorylates tyrosyl phosphorylated KRAS. a, b HEK293 cells were transfected with the indicated plasmids. Cells were lysed, immunoprecipitated, and immunoblotted with the indicated antibodies. c Phosphorylation was initiated by addition of 2 μM of Src to 250 μM of KRAS in the presence of 2 mM ATP and 1 mM activated sodium vanadate. Sequential ¹H-¹⁵N HSQC NMR spectra were collected, and peaks from un-phosphorylated and phosphorylated Tyr32 (blue) and Tyr64 (red) were integrated from each spectrum to plot curves of the fraction phosphorylated vs. time for each site. The error bars correspond to the noise-to-signal ratio of the respective peaks. The relative rates of phosphorylation of each site determined from a representative experiment (performed in duplicate) by curve fitting are shown in histograms on the right. The error bars indicate the standard curve fitting error obtained from the fitting analysis. d SHP2 (2 μM) was added to Src-phosphorylated KRAS sample purified in the presence of vanadate, and sequential ¹H-¹⁵N NMR spectra were collected to monitor dephosphorylation. Similar to c, the fractions of phosphorylated Tyr32 (blue) and Tyr64 (red) were plotted and rates of dephosphorylation were determined and are shown in the histograms. Rate curves from a representative experiment (performed in duplicate) were fitted to one phase exponential decay/association functions using the GraphPad Prism 4.0 software. Histogram error bars indicate the standard curve fitting error obtained from the fitting analysis. e Top panel; experimental set-up whereby two tubes containing 200 μM of KRAS and 3 μM of Src were incubated in in the presence of 2 mM ATP for 2 h (in the absence of vanadate). Sample 1 was then snap frozen and stored at −80 °C for MS analysis, and 3 μM of SHP2 was added to the remaining tube and incubated for an additional 2 h. Sample 2 was then frozen. Bottom panels; MS spectra of samples 1 and 2 with phosphorylation state of each mass indicated. f HEK293 cells transfected with the indicated plasmids were treated with (+) or without (−) 10 μM of 11a-1, lysed, immunoprecipitated, and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three independent experiments