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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 Ser1039 and Thr1041 by LC-MS/MS and SRM. A and B, trypsin-digested immuno (anti-FLAG) purified EGFR proteins were analyzed by high resolution LC-MS/MS. MS/MS spectra of the EGFR peptide TPLLSSLpS1039ATSNNSTVACIDR (where pS is phosphoserine) (A) and TPLLSSLSApT1041SNNSTVACIDR (where pT is phosphothreonine) (B) are shown. The y and b series ions are indicated. Arrows point to product y12 ions. As expected the m/z values of y12 ions are different between Ser(P)1039 (A) and Thr(P)1041 (B) peptides because Thr1041 or Thr(P)1041 is the amino-terminal residue in the y12 ions. C, the orbitrap XIC for the isobaric phosphopeptides with m/z 1144.04 and containing Ser(P)1039 or Thr(P)1041, as indicated, are shown to be chromatographically resolved. D, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated TPLLSSLSATSNNSTVACIDR (precursor, m/z = 1144.04, z = 2) transitioning to 1) precursor − 98/2 (m/z = 1095.05, z = 2) and 2) y5 (m/z = 634.30, z = 1) as indicated. E and F, histograms depicting the integrated SRM signal intensities of Ser(P)1039 peptide (E) and Thr(P)1041 peptide (F) normalized to a control EGFR peptide in the same sample (see “Experimental Procedures”). EGFR, Y998F, and S991A cell cultures were treated without (Control) or with (+EGF) EGF (100 ng/ml, 15 min, 37 °C) or with EGF after a 30-min pretreatment with the p38 inhibitor SB-202190 (10 μm) (+EGF+SB). Error bars indicate S.D. from three biological repeats of the experiment. For statistical analysis, Y998F and S991A samples were compared with wild type EGFR in EGF-stimulated samples (+EGF), and EGFR, Y998F and S991A samples in SB-202190 treated cells (+EGF+SB) were compared with themselves in EGF-stimulated samples (+EGF). *, p = 0.05 compared with wild type EGFR +EGF. In all cases phosphopeptides were statistically significantly less (p = 0.05) for control and SB-202190-treated samples compared with cognate +EGF samples. XIC, extracted ion current.
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Figure 7: Detection and quantification of phosphorylation at EGFR Ser1039 and Thr1041 by LC-MS/MS and SRM. A and B, trypsin-digested immuno (anti-FLAG) purified EGFR proteins were analyzed by high resolution LC-MS/MS. MS/MS spectra of the EGFR peptide TPLLSSLpS1039ATSNNSTVACIDR (where pS is phosphoserine) (A) and TPLLSSLSApT1041SNNSTVACIDR (where pT is phosphothreonine) (B) are shown. The y and b series ions are indicated. Arrows point to product y12 ions. As expected the m/z values of y12 ions are different between Ser(P)1039 (A) and Thr(P)1041 (B) peptides because Thr1041 or Thr(P)1041 is the amino-terminal residue in the y12 ions. C, the orbitrap XIC for the isobaric phosphopeptides with m/z 1144.04 and containing Ser(P)1039 or Thr(P)1041, as indicated, are shown to be chromatographically resolved. D, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated TPLLSSLSATSNNSTVACIDR (precursor, m/z = 1144.04, z = 2) transitioning to 1) precursor − 98/2 (m/z = 1095.05, z = 2) and 2) y5 (m/z = 634.30, z = 1) as indicated. E and F, histograms depicting the integrated SRM signal intensities of Ser(P)1039 peptide (E) and Thr(P)1041 peptide (F) normalized to a control EGFR peptide in the same sample (see “Experimental Procedures”). EGFR, Y998F, and S991A cell cultures were treated without (Control) or with (+EGF) EGF (100 ng/ml, 15 min, 37 °C) or with EGF after a 30-min pretreatment with the p38 inhibitor SB-202190 (10 μm) (+EGF+SB). Error bars indicate S.D. from three biological repeats of the experiment. For statistical analysis, Y998F and S991A samples were compared with wild type EGFR in EGF-stimulated samples (+EGF), and EGFR, Y998F and S991A samples in SB-202190 treated cells (+EGF+SB) were compared with themselves in EGF-stimulated samples (+EGF). *, p = 0.05 compared with wild type EGFR +EGF. In all cases phosphopeptides were statistically significantly less (p = 0.05) for control and SB-202190-treated samples compared with cognate +EGF samples. XIC, extracted ion current.

Mentions: The monophosphorylated isobaric isomers containing either Ser(P)1039 or Thr(P)1041 were analyzed by LC-MS/MS, which confirmed their identities. For example, their y12 fragment ions were clearly distinctive (Fig. 7, A and B). The two species did not co-elute during reverse phase chromatography, and this facilitated their resolution and ion current measurement by high resolution LC-MS (Fig. 7C). To facilitate the quantification of these species by SRM, the measurement of transitions involving distinctive y ions was attempted but proved inefficient. Therefore, an optimized SRM method was developed that included neutral loss of phosphoric acid (i.e. doubly charged parent − 98/2) and y5-associated transitions, which were common to the two phosphoisomers and efficiently detected (Fig. 7, A, B, and D). Note that different elution gradients were used for the LC-MS and SRM analyses shown in Fig. 7, C and D, respectively, so the retention times were not identical. The Orbitrap MS extracted ion current chromatograph and triple quadrupole SRM traces were similar (Fig. 7, compare C and D), and several repeats of the LC-MS/MS experiment indicated that the Ser(P)1039 peptide consistently eluted immediately before the Thr(P)1041 isomer.


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 Ser1039 and Thr1041 by LC-MS/MS and SRM. A and B, trypsin-digested immuno (anti-FLAG) purified EGFR proteins were analyzed by high resolution LC-MS/MS. MS/MS spectra of the EGFR peptide TPLLSSLpS1039ATSNNSTVACIDR (where pS is phosphoserine) (A) and TPLLSSLSApT1041SNNSTVACIDR (where pT is phosphothreonine) (B) are shown. The y and b series ions are indicated. Arrows point to product y12 ions. As expected the m/z values of y12 ions are different between Ser(P)1039 (A) and Thr(P)1041 (B) peptides because Thr1041 or Thr(P)1041 is the amino-terminal residue in the y12 ions. C, the orbitrap XIC for the isobaric phosphopeptides with m/z 1144.04 and containing Ser(P)1039 or Thr(P)1041, as indicated, are shown to be chromatographically resolved. D, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated TPLLSSLSATSNNSTVACIDR (precursor, m/z = 1144.04, z = 2) transitioning to 1) precursor − 98/2 (m/z = 1095.05, z = 2) and 2) y5 (m/z = 634.30, z = 1) as indicated. E and F, histograms depicting the integrated SRM signal intensities of Ser(P)1039 peptide (E) and Thr(P)1041 peptide (F) normalized to a control EGFR peptide in the same sample (see “Experimental Procedures”). EGFR, Y998F, and S991A cell cultures were treated without (Control) or with (+EGF) EGF (100 ng/ml, 15 min, 37 °C) or with EGF after a 30-min pretreatment with the p38 inhibitor SB-202190 (10 μm) (+EGF+SB). Error bars indicate S.D. from three biological repeats of the experiment. For statistical analysis, Y998F and S991A samples were compared with wild type EGFR in EGF-stimulated samples (+EGF), and EGFR, Y998F and S991A samples in SB-202190 treated cells (+EGF+SB) were compared with themselves in EGF-stimulated samples (+EGF). *, p = 0.05 compared with wild type EGFR +EGF. In all cases phosphopeptides were statistically significantly less (p = 0.05) for control and SB-202190-treated samples compared with cognate +EGF samples. XIC, extracted ion current.
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Figure 7: Detection and quantification of phosphorylation at EGFR Ser1039 and Thr1041 by LC-MS/MS and SRM. A and B, trypsin-digested immuno (anti-FLAG) purified EGFR proteins were analyzed by high resolution LC-MS/MS. MS/MS spectra of the EGFR peptide TPLLSSLpS1039ATSNNSTVACIDR (where pS is phosphoserine) (A) and TPLLSSLSApT1041SNNSTVACIDR (where pT is phosphothreonine) (B) are shown. The y and b series ions are indicated. Arrows point to product y12 ions. As expected the m/z values of y12 ions are different between Ser(P)1039 (A) and Thr(P)1041 (B) peptides because Thr1041 or Thr(P)1041 is the amino-terminal residue in the y12 ions. C, the orbitrap XIC for the isobaric phosphopeptides with m/z 1144.04 and containing Ser(P)1039 or Thr(P)1041, as indicated, are shown to be chromatographically resolved. D, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated TPLLSSLSATSNNSTVACIDR (precursor, m/z = 1144.04, z = 2) transitioning to 1) precursor − 98/2 (m/z = 1095.05, z = 2) and 2) y5 (m/z = 634.30, z = 1) as indicated. E and F, histograms depicting the integrated SRM signal intensities of Ser(P)1039 peptide (E) and Thr(P)1041 peptide (F) normalized to a control EGFR peptide in the same sample (see “Experimental Procedures”). EGFR, Y998F, and S991A cell cultures were treated without (Control) or with (+EGF) EGF (100 ng/ml, 15 min, 37 °C) or with EGF after a 30-min pretreatment with the p38 inhibitor SB-202190 (10 μm) (+EGF+SB). Error bars indicate S.D. from three biological repeats of the experiment. For statistical analysis, Y998F and S991A samples were compared with wild type EGFR in EGF-stimulated samples (+EGF), and EGFR, Y998F and S991A samples in SB-202190 treated cells (+EGF+SB) were compared with themselves in EGF-stimulated samples (+EGF). *, p = 0.05 compared with wild type EGFR +EGF. In all cases phosphopeptides were statistically significantly less (p = 0.05) for control and SB-202190-treated samples compared with cognate +EGF samples. XIC, extracted ion current.
Mentions: The monophosphorylated isobaric isomers containing either Ser(P)1039 or Thr(P)1041 were analyzed by LC-MS/MS, which confirmed their identities. For example, their y12 fragment ions were clearly distinctive (Fig. 7, A and B). The two species did not co-elute during reverse phase chromatography, and this facilitated their resolution and ion current measurement by high resolution LC-MS (Fig. 7C). To facilitate the quantification of these species by SRM, the measurement of transitions involving distinctive y ions was attempted but proved inefficient. Therefore, an optimized SRM method was developed that included neutral loss of phosphoric acid (i.e. doubly charged parent − 98/2) and y5-associated transitions, which were common to the two phosphoisomers and efficiently detected (Fig. 7, A, B, and D). Note that different elution gradients were used for the LC-MS and SRM analyses shown in Fig. 7, C and D, respectively, so the retention times were not identical. The Orbitrap MS extracted ion current chromatograph and triple quadrupole SRM traces were similar (Fig. 7, compare C and D), and several repeats of the LC-MS/MS experiment indicated that the Ser(P)1039 peptide consistently eluted immediately before the Thr(P)1041 isomer.

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