Fig. 3: KRAS Q61H is phosphorylated by Src and dephosphorylated by SHP2.

a HEK293 cells were transfected with HA-tagged KRAS WT, G12D, G12V, or Q61H and either kinase-dead (KD) or active (WT) Src. KRAS was immunoprecipitated with anti-HA antibody, resolved on SDS-PAGE, and immunoblotted with anti-pTyr. Equal amounts of lysates were resolved on SDS-PAGE and immunoblotted with an antibody recognizing phosphorylated KRAS Y64, as well as the antibodies indicated to confirm expression and loading. b KRAS Q61H loaded with GDP or a non-hydrolysable GTP-analog (GMPPNP) was incubated with Src (1 : 250 Src to KRAS ratio) and ATP, and the reaction products were analyzed with mass spectrometry. The predicted mass of ¹⁵N-KRAS Q61H (residues 1–173 C118S) is 20084.36. Peaks corresponding to unmodified KRAS Q61H, as well as KRAS Q61H phosphorylated at one (+80 Da) and two sites (+160 Da) are labeled accordingly. c KRAS4B Q61H sequence indicating tryptic peptides in which phosphotyrosine was detected (orange letters with blue arrows indicating trypsin cleavage sites). d, e LC-MS analyses of phosphorylated tyrosine residues in KRAS Q61H. Two phosphorylated tyrosine residues from the KRAS Q61H protein sequence were identified from in vitro kinase reaction samples that were reduced/alkylated, trypsin-digested, and analyzed by LC-MS. MS/MS spectra matched KRAS tryptic peptides containing phosphorylated d Y32 and e Y64. f HEK293 cells were transfected with HA-KRAS WT, Q61H, Y32F/Q61H, or Q61H/Y64F mutants, with or without co-transfection of Src. HA-KRAS was immunoprecipitated with anti-HA and probed with anti-pTyr antibody. The whole-cell extracts were resolved on SDS-PAGE and immunoblotted with an antibody recognizing KRAS pTyr64, as well as those indicated, to confirm expression and loading. g HEK293 cells were transfected with HA-KRAS WT or Q61H increasing amounts of SHP2, with or without co-transfection of Src. HA-KRAS was immunoprecipitated with HA antibody and blotted with pTyr antibody. The whole-cell extracts (WCEs) were blotted with the antibodies indicated to confirm expression and loading. All immunoblot data are representative of at least three independent experiments. h Src-phosphorylated KRAS Q61H was incubated with SHP2 for 2 h and the total mass was analyzed using LC/MS as explained in “Methods.” The predicted mass of unmodified ¹⁵N-KRAS Q61H (residues 1–173 C118S) is 20084.36. i CRISPR/Cas9-mediated non-target (SHP2+/+) or SHP2−/− Hs766T cells were serum-starved for 20 h and and treated with (200 ng/ml) (+) or without (−) EGF for 1.5 min. One milligram of lysates was immunoprecipitated and immunoblotted with the indicated antibodies. a, f, g, i The blots are representative of three independent experiments.