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Epidermal growth factor receptor phosphorylation sites Ser991 and Tyr998 are implicated in the regulation of receptor endocytosis and phosphorylations at Ser1039 and Thr1041.

Tong J, Taylor P, Peterman SM, Prakash A, Moran MF - Mol. Cell Proteomics (2009)

Bottom Line: Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin.The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041).These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Structure and Function, The Hospital For Sick Children, and The McLaughlin Centre For Molecular Medicine, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser(991) and Tyr(998) accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041). These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195-4206). EGF-induced phosphorylations at Ser(1039) and Thr(1041) were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

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Detection and quantification of phosphorylation at EGFR Ser991 by SRM. A, trypsin-digested immunopurified EGFR was analyzed by high resolution LC-MS/MS (Proxeon LC/source; Thermo LTQ-Orbitrap). The MS/MS spectrum of the EGFR peptide MHLPpS991PTDSNFYR (where pS is phosphoserine) is shown (upper panel). The y and b series ions are indicated. Arrows point to product ions corresponding to parent − 98 and y10 −98 ions (as indicated), which reflect the neutral loss of phosphoric acid from the Ser(P) side chain. B, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated MHLPSPTDSNFYR (precursor, m/z = 822.85, z = 2) transitioning to 1) precursor − 98/2 (m/z = 773.86, z = 2) and 2) y10 − 98 (m/z = 1165.50, z = 1) at various time points after EGF stimulation as indicated. C, histogram depicting the integrated SRM signal intensities normalized to a non-phosphorylated EGFR peptide in the same sample. Error bars indicate S.D. from three biological repeats of the experiment. D and E, Western blot (WB) analysis of whole-cell protein extracts from HEK-EGFR cells harvested following EGF treatment for the indicated durations. Antibodies specific to the EGFR phosphoepitope containing Tyr(P)1092 (D) or that recognize the FLAG epitope on EGFR-FLAG protein (E) were used to probe the filters, which were further developed with appropriate secondary antibody-enzyme conjugates and chemiluminescence imaging (see “Experimental Procedures”). pY, phosphotyrosine.
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Figure 1: Detection and quantification of phosphorylation at EGFR Ser991 by SRM. A, trypsin-digested immunopurified EGFR was analyzed by high resolution LC-MS/MS (Proxeon LC/source; Thermo LTQ-Orbitrap). The MS/MS spectrum of the EGFR peptide MHLPpS991PTDSNFYR (where pS is phosphoserine) is shown (upper panel). The y and b series ions are indicated. Arrows point to product ions corresponding to parent − 98 and y10 −98 ions (as indicated), which reflect the neutral loss of phosphoric acid from the Ser(P) side chain. B, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated MHLPSPTDSNFYR (precursor, m/z = 822.85, z = 2) transitioning to 1) precursor − 98/2 (m/z = 773.86, z = 2) and 2) y10 − 98 (m/z = 1165.50, z = 1) at various time points after EGF stimulation as indicated. C, histogram depicting the integrated SRM signal intensities normalized to a non-phosphorylated EGFR peptide in the same sample. Error bars indicate S.D. from three biological repeats of the experiment. D and E, Western blot (WB) analysis of whole-cell protein extracts from HEK-EGFR cells harvested following EGF treatment for the indicated durations. Antibodies specific to the EGFR phosphoepitope containing Tyr(P)1092 (D) or that recognize the FLAG epitope on EGFR-FLAG protein (E) were used to probe the filters, which were further developed with appropriate secondary antibody-enzyme conjugates and chemiluminescence imaging (see “Experimental Procedures”). pY, phosphotyrosine.

Mentions: SRM intensities for associated proteins were divided by the value measured for the EGFR normalization peptide YSFGATCVK (residues 285–293; see “Results”) intrinsic to that sample. Hence calculated associated protein amounts are per unit of EGFR. The assumption was made that the amount of EGFR in cells did not change significantly after 15 min of EGF stimulation or SB-202190 incubation. Western blot results verified this assumption (e.g. Fig. 1E). Results from three independently repeated experiments were used to calculate statistical significance by the Student's t test. A complete listing of SRM intensities including coefficients of variance are included in the supplemental information.


Epidermal growth factor receptor phosphorylation sites Ser991 and Tyr998 are implicated in the regulation of receptor endocytosis and phosphorylations at Ser1039 and Thr1041.

Tong J, Taylor P, Peterman SM, Prakash A, Moran MF - Mol. Cell Proteomics (2009)

Detection and quantification of phosphorylation at EGFR Ser991 by SRM. A, trypsin-digested immunopurified EGFR was analyzed by high resolution LC-MS/MS (Proxeon LC/source; Thermo LTQ-Orbitrap). The MS/MS spectrum of the EGFR peptide MHLPpS991PTDSNFYR (where pS is phosphoserine) is shown (upper panel). The y and b series ions are indicated. Arrows point to product ions corresponding to parent − 98 and y10 −98 ions (as indicated), which reflect the neutral loss of phosphoric acid from the Ser(P) side chain. B, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated MHLPSPTDSNFYR (precursor, m/z = 822.85, z = 2) transitioning to 1) precursor − 98/2 (m/z = 773.86, z = 2) and 2) y10 − 98 (m/z = 1165.50, z = 1) at various time points after EGF stimulation as indicated. C, histogram depicting the integrated SRM signal intensities normalized to a non-phosphorylated EGFR peptide in the same sample. Error bars indicate S.D. from three biological repeats of the experiment. D and E, Western blot (WB) analysis of whole-cell protein extracts from HEK-EGFR cells harvested following EGF treatment for the indicated durations. Antibodies specific to the EGFR phosphoepitope containing Tyr(P)1092 (D) or that recognize the FLAG epitope on EGFR-FLAG protein (E) were used to probe the filters, which were further developed with appropriate secondary antibody-enzyme conjugates and chemiluminescence imaging (see “Experimental Procedures”). pY, phosphotyrosine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742444&req=5

Figure 1: Detection and quantification of phosphorylation at EGFR Ser991 by SRM. A, trypsin-digested immunopurified EGFR was analyzed by high resolution LC-MS/MS (Proxeon LC/source; Thermo LTQ-Orbitrap). The MS/MS spectrum of the EGFR peptide MHLPpS991PTDSNFYR (where pS is phosphoserine) is shown (upper panel). The y and b series ions are indicated. Arrows point to product ions corresponding to parent − 98 and y10 −98 ions (as indicated), which reflect the neutral loss of phosphoric acid from the Ser(P) side chain. B, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated MHLPSPTDSNFYR (precursor, m/z = 822.85, z = 2) transitioning to 1) precursor − 98/2 (m/z = 773.86, z = 2) and 2) y10 − 98 (m/z = 1165.50, z = 1) at various time points after EGF stimulation as indicated. C, histogram depicting the integrated SRM signal intensities normalized to a non-phosphorylated EGFR peptide in the same sample. Error bars indicate S.D. from three biological repeats of the experiment. D and E, Western blot (WB) analysis of whole-cell protein extracts from HEK-EGFR cells harvested following EGF treatment for the indicated durations. Antibodies specific to the EGFR phosphoepitope containing Tyr(P)1092 (D) or that recognize the FLAG epitope on EGFR-FLAG protein (E) were used to probe the filters, which were further developed with appropriate secondary antibody-enzyme conjugates and chemiluminescence imaging (see “Experimental Procedures”). pY, phosphotyrosine.
Mentions: SRM intensities for associated proteins were divided by the value measured for the EGFR normalization peptide YSFGATCVK (residues 285–293; see “Results”) intrinsic to that sample. Hence calculated associated protein amounts are per unit of EGFR. The assumption was made that the amount of EGFR in cells did not change significantly after 15 min of EGF stimulation or SB-202190 incubation. Western blot results verified this assumption (e.g. Fig. 1E). Results from three independently repeated experiments were used to calculate statistical significance by the Student's t test. A complete listing of SRM intensities including coefficients of variance are included in the supplemental information.

Bottom Line: Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin.The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041).These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Structure and Function, The Hospital For Sick Children, and The McLaughlin Centre For Molecular Medicine, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser(991) and Tyr(998) accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041). These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195-4206). EGF-induced phosphorylations at Ser(1039) and Thr(1041) were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

Show MeSH
Related in: MedlinePlus