Figure 3: WHSC1L1-mediated mono-methylation of EGFR at lysine K721 enhances activating phosphorylation marks of EGFR independently of EGF stimulation and WHSC1L1 knockdown decreases phospho-EGFR and phospho-ERK in SCCHN cells.

(a) 293T cells were cotransfected with HA-WHSC1L1 and FLAG-EGFR-WT versus FLAG-EGFR-K721A. After 48 h of serum starvation and EGF stimulation, FLAG-immunoprecipitates were blotted for phospho-EGFR marks. Densitometry of immunoblots from this experiment is also shown. (b) 293T cells were cotransfected with HA-WHSC1L1 and FLAG-EGFR-WT, HA-WHSC1L1 and FLAG-EGFR-K721A, or an enzyme dead variant of WHSC1L1 (HA-WHSC1L1 1-1073) and FLAG-EGFR-WT. Cells were serum starved for 48 h and FLAG-immunoprecipitates without EGF stimulation were blotted for phospho-EGFR marks. Densitometry of immunoblots from this experiment is also shown. (c) 3D structural representation of the tyrosine kinase domain of EGFR in its “DFG-in” form (Protein Data Bank, entry 2GS7). (d) Effect of WHSC1L1 (nuclear extract) knockdown on phospho-EGFR marks, EGFR K721 mono-methylation and phospho-ERK1/2 levels (cytoplasmic extract) in YD-10B cells assessed by Western blotting. Actin and H3 were blotted as loading controls. Densitometry of immunoblots from each experiment is also shown. Data were obtained from two separate experiments. (e) Effect of WHSC1L1 (nuclear extract) knockdown on phospho-EGFR marks, EGFRK721 mono-methylation and phospho-ERK1/2 levels (cytoplasmic extract) in HN13 cells assessed by Western blotting. Actin and H3 were blotted as loading controls. Densitometry of immunoblots from this experiment is also shown. Data were obtained from the same experiment.