Figure 1: WHSC1L1 methylates EGFR at lysine K721 in vitro and in vivo.
(a) WHSC1L1 methylates EGFR in a dose-dependent manner. In vitro methyltransferase assay of WHSC1L1 with recombinant EGFR. Recombinant EGFR was incubated with increasing amounts of WHSC1L1 in the presence of 3H-SAM. Bovine serum albumin was used as a negative control and recombinant histone H3 as a positive control. The reactants were analyzed by SDS-PAGE followed by fluorography for 3 days (upper panel). The PVDF transfer membrane was then stained with MemCode reversible protein stain to visualize the total protein (lower panel). (b) The MS/MS spectrum corresponding to the mono-methylated EGFR fragment IPVAIKEL (716-723). The mono-methylation corresponds to lysine (K) at position 721 within the tyrosine kinase domain of EGFR. MS/MS score, Mascot ion score and Expectation value in Mascot Database search results are shown. (c) Aminoacid sequence alignment of human EGFR. The IPVAIKEL sequence which includes lysine K721 is located within the tyrosine kinase domain of EGFR and is preserved from Homo sapiens to Xenopus laevis. (d) Evaluation of the specificity of anti-mono-methylated K721 EGFR antibody using enzyme-linked immunosorbent assay (ELISA). Y-axis represents ELISA optical density (OD) units read at 492 nm. ELISA plates coated with the mono-methylated K721 EGFR peptide versus the unmodified EGFR peptide were incubated with the primary rabbit antisera for 16 h at 4oC. Detection was performed using a secondary rabbit antibody conjugated with horseradish peroxidase. Primary rabbit antisera were examined after dual selection against the modified versus the unmodified peptides. (e) 293T cells were cotransfected with FLAG-EGFR-WT or FLAG-EGFR-K721A and HA-Mock or HA-WHSC1L1. Immunoprecipitation with an anti-FLAG antibody was performed and immunoprecipitates were blotted with anti-mono-methylated K721 EGFR, anti-FLAG and anti-HA antibodies.
