Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation

Abstract

Epidermal growth factor receptor (EGFR) can undergo post-translational modifications, including phosphorylation, glycosylation and ubiquitylation, leading to diverse physiological consequences and modulation of its biological activity. There is increasing evidence that methylation may parallel other post-translational modifications in the regulation of various biological processes. It is still not known, however, whether EGFR is regulated by this post-translational event. Here, we show that EGFR Arg 1175 is methylated by an arginine methyltransferase, PRMT5. Arg 1175 methylation positively modulates EGF-induced EGFR trans-autophosphorylation at Tyr 1173, which governs ERK activation. Abolishment of Arg 1175 methylation enhances EGF-stimulated ERK activation by reducing SHP1 recruitment to EGFR, resulting in augmented cell proliferation, migration and invasion of EGFR-expressing cells. Therefore, we propose a model in which the regulatory crosstalk between PRMT5-mediated Arg 1175 methylation and EGF-induced Tyr 1173 phosphorylation attenuates EGFR-mediated ERK activation.

Figure 1

EGFR Arg 1175 is monomethylated. (a) In vivo methylation of EGFR. A431 cells were metabolically labelled with l-[methyl-³H] methionine (left) or l-[³⁵S] methionine (right) the presence or absence of protein synthesis inhibitors, as indicated. Immunoprecipitates (IP) of EGFR or control antibodies from l-[methyl-³H methionine-labelled cells were analysed by fluorography (lanes 1 and 2), Coomassie Blue staining (lanes 3 and 4) or western blotting with EGFR antibody (lanes 5 and 6). Whole-cell lysates of l-[³⁵S]methionine-labelled cells were analysed by Coomassie Blue staining (lanes 7 and 8) or autoradiography (lanes 9 and 10). (b) Mass spectrometry analysis of endogenous EGFR immunopurified from A431 cells. (c) Amino acid sequence of peptides corresponding to the EGFR 1171–1182 region, in which Arg 1175 is unmodified, monomethylated or dimethylated. Different amounts of peptides were spotted on PVDF membranes and detected by anti-EGFR or anti-EGFR methylated-Arg 1175 (me-Arg 1175) antibodies. (d) Western blot analysis of exogenous EGFR in HEK293 cells transfected with control vector, EGFR (WT) or EGFR (R1175K). (e) Western blot analysis of exogenous EGFR in HEK293 cells transfected with empty vector, EGFR (WT) or EGFR (R1175K). Anti-EGFR methylated-Arg 1175 antibody was pre-incubated with peptides, as indicated before use. (f) Western blot analysis of endogenous EGFR in MDA-MB-468 cells transfected with control or EGFR siRNAs. (g) Confocal microscopy analysis of MDA-MB-468 cells stained with total endogenous EGFR (red), methylated-Arg 1175 (green) and DAPI (blue). The third columns shows higher-magnification images of the areas outlined in the second column.

Figure 2

PRMT5 interacts with EGFR and methylates Arg 1175. (a) Western blot analysis of exogenous EGFR and PRMTs in the input and anti-EGFR immunoprecipitates from HEK293 cells transfected with EGFR and GFP–PRMTs, as indicated. (b) In vitro methylation assay of unmodified EGFR peptide by immunopurified HA–PRMT3, 5 or 8. Methylation of peptides was detected by western blotting (top) and scintillation counting (bottom). Error bars represent s.d. (n = 3). (c) Confocal microscopy analysis of MDA-MB-468 cells stained with endogenous EGFR (red), PRMT5 (green) and DAPI (blue). (d) Western blot analysis of endogenous EGFR and total PRMT5 of the MDA-MB-468 cells in which endogenous PRMT5 was knocked down by three PRMT5 siRNAs (lanes 1–4) and then rescued with an siRNA-resistant PRMT5 mutant (RR-PRMT5 ; lane 5).

Figure 3

Suppression of Arg 1175 methylation promotes EGFR-mediated cell proliferation, migration and invasion. (a) Left: western blot analysis of MCF7 stable transfectants expressing EGFR (WT), EGFR (R1175K) or empty vector. Right: in vitro cell proliferation rates were assayed using the MTT colorimetric method. Error bars represent s.d. (n = 5). (b) Left: in vivo cell proliferation was measured using an orthotopic breast cancer mouse model. Error bars represent s.d. (n = 10). Right: two representative tumours from each group in the sixth week after inoculation. (c) Migration assay of these stable transfectants. Statistical analysis was carried out using Student’s t -test. Error bars represent s.d. (n = 3). (d) Invasion assay of these stable transfectants. Statistical analysis was carried out using Student’s t -test. Error bars represent s.d. (n = 3).

Figure 4

Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation. (a) Top: western blot analysis of exogenous EGFR in EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of phospho-EGFR Tyr 1173 (p-Tyr 1173) blot. Error bars represent s.d. (n 3). (b) Top: western blot analysis of endogenous EGFR in EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of phospho-EGFR Tyr 1173 (p-Tyr 1173) blot. Error bars represent s.d. (n = 3). (c) In vitro kinase assay of unmodified (open symbols) and monomethylated (filled symbols) EGFR peptides by immunopurified EGFR proteins. Phosphorylation of peptides was detected by western blotting using anti-EGFR phospho-Tyr 1173 antibody (left) and scintillation counting (right). Error bars represent s.d. (n = 3).

Figure 5

Suppression of Arg 1175 methylation inhibits SHP1 recruitment and prolongs ERK activation. (a) Top: western blot analysis of EGFR, SHP1, Grb2 and SHC in the input and anti-EGFR immunoprecipitates from EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of EGFR-bound SHP1 blot. Error bars represent s.d. (n 3). (b) Top: western blot analysis of endogenous EGFR, PRMT5, SHP1, Grb2=and SHC in the input and anti-EGFR immunoprecipitates from EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of EGFR-bound SHP1 blot. Error bars represent s.d. (n 3). (c) Top: western blot analysis of endogenous ERK, PLC-γ , STAT3 and AKT in EGF-stimulated MCF7-EGFR (WT) and MCF7-EGFR (R1175K) stable transfectants. Bottom: densitometry of phospho-ERK (p-ERK) blot. Error bars represent s.d. (n = 3). (d) Top: western blot analysis of endogenous EGFR, PRMT5, ERK, PLC-γ , STAT3 and AKT in EGF-stimulated MDA-MB-468 cells transfected with control or PRMT5 siRNA #1. Bottom: densitometry of phospho-ERK (p-ERK) blot. Error bars represent s.d. (n = 3).