Fig. 5
From: Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation
Tyrosyl phosphorylation of KRAS disrupts GTPase cycle. a Overlay of four 1H-15N HSQC spectra of two representative residues T74 and M67, in which the four peaks represent GDP-loaded KRAS (black), GTP-loaded KRAS (blue), GDP-loaded Src-phosphorylated (p)KRAS (red), and GTP-loaded pKRAS (green). b Real-time NMR-derived nucleotide exchange curves for unmodified vs. Src-phosphorylated KRAS. A 250μM sample of GDP-loaded 15N KRAS (black) or pKRAS (blue) was incubated with ten-fold molar excess of GTPγS. Each dot represents mean fraction of KRAS that is loaded with GDP on the basis of peak intensities [IGDP/(IGDP + IGTPɣS)] from the same three residues of KRAS and pKRAS. SOScat was added at a ratio of 1:600 to KRAS (orange) or pKRAS (red). c Src phosphorylation of KRAS impairs SOScat-assisted nucleotide exchange. Fold increase in the rate of exchange upon addition of SOScat to KRAS or pKRAS. The reaction was performed twice in the absence and twice in the presence of SOScat and four ratios were determined from each pairwise comparison. d GTP hydrolysis curves illustrating intrinsic and RASA1 GAP domain (1:3000 ratio) assisted GTP hydrolysis for unmodified KRAS and pKRAS. e Src phosphorylation of KRAS reduces sensitivity to GAP activity. Fold increase in the rate of GTP hydrolysis with addition of RASA1 GAP domain for KRAS vs. pKRAS. The reaction was performed twice in the absence and twice in the presence of RASA1 GAP domain and four ratios were determined from each pairwise comparison. b, d present a single representative experiment that was repeated at least twice. Error bars represent the standard deviation of the fraction GDP as reported by three pairs of cross-peaks. Error bars in c, e indicate standard deviation of the fold changes
