Reactants were separated on SDS-PAGE and stained with Simply Blue Safe Stain (Life Technologies)
September 22, 2021
Reactants were separated on SDS-PAGE and stained with Simply Blue Safe Stain (Life Technologies). malignancy pathogenesis through promotion of cell proliferation, invasion, migration, metastasis and angiogenesis1,2,3,4. It is primarily a cytoplasmic-membrane RTK activated by growth factor ligands, such as epidermal growth factor and amphiregulin, which induce homodimerization and/or heterodimerization of EGFR with other members of the same RTK family (HER2, HER3, HER4). This dimerization increases the intrinsic tyrosine kinase activity and subsequent autophosphorylation of C-terminal tyrosine (Y) residues, such as Y992, Y1068, Y1148, and Y1173 of EGFR. These phosphotyrosines function as docking sites for SH2-made up of messenger HA-100 dihydrochloride molecules, which in turn activate the downstream pathways of RAS-RAF-MEK-ERK, PI3K-AKT-mTOR and STAT3, leading to DNA synthesis and cell proliferation, invasion and migration5,6,7,8. Post-translational modifications of EGFR, such as phosphorylation, glycosylation and ubiquitination, are known to modulate the function of EGFR6,7,9,10,11,12. No reports though have described the functions of EGFR lysine methylation. More recently, EGFR arginine (R) HA-100 dihydrochloride methylation was reported to play significant functions in the regulation of EGFR function. Hsu and through H3K36 di-methylation in SCCHN cells29, we further attempted to assess whether HA-100 dihydrochloride the oncogenic activity of WHSC1L1 is also mediated through a non-histone protein substrate(s). In this study, we show that WHSC1L1 mono-methylates lysine 721 in the tyrosine kinase domain name of EGFR, and that this methylation enhances activation of its downstream RAS-RAF-MEK-ERK cascade, even without epidermal growth factor stimulation. We also show that WHSC1L1-mediated EGFR mono-methylation affects the function of nuclear EGFR by enhancing its conversation with PCNA, and that this may be a novel mechanism to enhance DNA synthesis and S phase progression. These findings may have significant clinical implications and provide the scientific rationale for further investigation of WHSC1L1 inhibition as a novel HA-100 dihydrochloride treatment approach in patients with SCCHN. Results WHSC1L1 mono-methylates EGFR at lysine K721 in its tyrosine kinase domain name both and methyltransferase assays using a proprietary library of recombinant oncogenic or tumor-suppressor proteins that are known to be important in SCCHN oncogenesis. This initial screening revealed EGFR as a potential substrate of WHSC1L1. methyltransferase assay with increasing amounts of WHSC1L1 revealed dose-dependent EGFR methylation (Fig. 1a), further supporting WHSC1L1-mediated EGFR methylation. In order to confirm the EGFR methylation by WHSC1L1 and to identify a methylated amino-acid residue(s), we performed mass spectrometry analysis of the methylated EGFR and found that WHSC1L1 mono-methylated lysine K721 in the tyrosine kinase domain name of EGFR (Fig. 1b). Given the high conservation of lysine K721 among various species from to (Fig. 1c), we presumed that mono-methylation of K721 may play a significant role in the functional regulation of EGFR. Open in a separate window Physique 1 WHSC1L1 methylates EGFR at lysine K721 and 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 name of EGFR. MS/MS score, Mascot ion score and Expectation CIP1 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 name of EGFR and is preserved from to and methylation of EGFR by WHSC1L1. No associations.