Limits...
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.

Show MeSH

Related in: MedlinePlus

SRM-based measurement of EGFR-associated proteins. EGFR was recovered by anti-FLAG IP from HEK cells stably expressing WT EGFR or one of the variants Y998F or S991A. The immune complexes were converted to tryptic peptides, which were subjected to SRM analysis to specifically monitor transitions derived from the indicated EGFR-associated proteins (see Table II). Protein amounts are expressed per unit of EGFR protein after normalization with an intrinsic EGFR peptide (see “Experimental Procedures”). Protein amounts are shown as EGF-induced -fold increases relative to unstimulated cells (means ± S.E., n = 3). For the purpose of constructing the histogram, values from unstimulated (i.e. no EGF) cells were set to unity; however, variation associated with all measurements were included in Student's t test calculations. Asterisks above bars indicate statistically significant differences from the amount of receptor-associated protein measured with unstimulated cells (*, p = 0.05; **, p = 0.01; ***, p = 0.005). Additionally the level of receptor association achieved by each of the proteins in EGF-stimulated cells was compared and found to be statistically different from WT (**, p = 0.01) only for CBL and ubiquitin (UBB) as indicated by the dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2742444&req=5

Figure 5: SRM-based measurement of EGFR-associated proteins. EGFR was recovered by anti-FLAG IP from HEK cells stably expressing WT EGFR or one of the variants Y998F or S991A. The immune complexes were converted to tryptic peptides, which were subjected to SRM analysis to specifically monitor transitions derived from the indicated EGFR-associated proteins (see Table II). Protein amounts are expressed per unit of EGFR protein after normalization with an intrinsic EGFR peptide (see “Experimental Procedures”). Protein amounts are shown as EGF-induced -fold increases relative to unstimulated cells (means ± S.E., n = 3). For the purpose of constructing the histogram, values from unstimulated (i.e. no EGF) cells were set to unity; however, variation associated with all measurements were included in Student's t test calculations. Asterisks above bars indicate statistically significant differences from the amount of receptor-associated protein measured with unstimulated cells (*, p = 0.05; **, p = 0.01; ***, p = 0.005). Additionally the level of receptor association achieved by each of the proteins in EGF-stimulated cells was compared and found to be statistically different from WT (**, p = 0.01) only for CBL and ubiquitin (UBB) as indicated by the dashed lines.

Mentions: To quantify the receptor-associated proteins, including changes due to EGF stimulation and as a consequence of the Y998F and S991A mutations, SRM analysis was applied to the receptor·immunoprecipitate complexes isolated from cells before and after EGF treatment. An SRM method that tracked the top six EGFR-associated proteins (according to spectral counts associated with WT EGFR; Table I) was formulated based on transitions summarized in supplemental Table 2. The fragmentation patterns obtained during discovery analyses with the LTQ-Orbitrap were a useful starting point toward the development of the SRM transitions. However, the resonance excitation-based fragmentations produced in the linear ion trap were not identical with the true CIDs undertaken in Q2 of the triple quadrupole TSQ instrument that was used for SRM. The intensity of b ions was generally greater in the linear ion trap, which also had a low molecular weight cutoff (i.e. ⅓ rule) that made low m/z ions more predominant in the triple quadrupole TSQ instrument. In addition, the LC gradients were of different durations (120 min with the Orbitrap and 40 min with the TSQ instrument). The SRM method was applied in three independent biological experiments resulting in the quantification the EGFR and the six receptor-associated proteins (Fig. 5 and Table II). The EGFR peptide YSFGATCVK (residues 285–293; described above), which is common to the WT and variant proteins and not subject to phosphorylation, was quantified and used to normalize for the amount of EGFR in each sample.


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)

SRM-based measurement of EGFR-associated proteins. EGFR was recovered by anti-FLAG IP from HEK cells stably expressing WT EGFR or one of the variants Y998F or S991A. The immune complexes were converted to tryptic peptides, which were subjected to SRM analysis to specifically monitor transitions derived from the indicated EGFR-associated proteins (see Table II). Protein amounts are expressed per unit of EGFR protein after normalization with an intrinsic EGFR peptide (see “Experimental Procedures”). Protein amounts are shown as EGF-induced -fold increases relative to unstimulated cells (means ± S.E., n = 3). For the purpose of constructing the histogram, values from unstimulated (i.e. no EGF) cells were set to unity; however, variation associated with all measurements were included in Student's t test calculations. Asterisks above bars indicate statistically significant differences from the amount of receptor-associated protein measured with unstimulated cells (*, p = 0.05; **, p = 0.01; ***, p = 0.005). Additionally the level of receptor association achieved by each of the proteins in EGF-stimulated cells was compared and found to be statistically different from WT (**, p = 0.01) only for CBL and ubiquitin (UBB) as indicated by the dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: SRM-based measurement of EGFR-associated proteins. EGFR was recovered by anti-FLAG IP from HEK cells stably expressing WT EGFR or one of the variants Y998F or S991A. The immune complexes were converted to tryptic peptides, which were subjected to SRM analysis to specifically monitor transitions derived from the indicated EGFR-associated proteins (see Table II). Protein amounts are expressed per unit of EGFR protein after normalization with an intrinsic EGFR peptide (see “Experimental Procedures”). Protein amounts are shown as EGF-induced -fold increases relative to unstimulated cells (means ± S.E., n = 3). For the purpose of constructing the histogram, values from unstimulated (i.e. no EGF) cells were set to unity; however, variation associated with all measurements were included in Student's t test calculations. Asterisks above bars indicate statistically significant differences from the amount of receptor-associated protein measured with unstimulated cells (*, p = 0.05; **, p = 0.01; ***, p = 0.005). Additionally the level of receptor association achieved by each of the proteins in EGF-stimulated cells was compared and found to be statistically different from WT (**, p = 0.01) only for CBL and ubiquitin (UBB) as indicated by the dashed lines.
Mentions: To quantify the receptor-associated proteins, including changes due to EGF stimulation and as a consequence of the Y998F and S991A mutations, SRM analysis was applied to the receptor·immunoprecipitate complexes isolated from cells before and after EGF treatment. An SRM method that tracked the top six EGFR-associated proteins (according to spectral counts associated with WT EGFR; Table I) was formulated based on transitions summarized in supplemental Table 2. The fragmentation patterns obtained during discovery analyses with the LTQ-Orbitrap were a useful starting point toward the development of the SRM transitions. However, the resonance excitation-based fragmentations produced in the linear ion trap were not identical with the true CIDs undertaken in Q2 of the triple quadrupole TSQ instrument that was used for SRM. The intensity of b ions was generally greater in the linear ion trap, which also had a low molecular weight cutoff (i.e. ⅓ rule) that made low m/z ions more predominant in the triple quadrupole TSQ instrument. In addition, the LC gradients were of different durations (120 min with the Orbitrap and 40 min with the TSQ instrument). The SRM method was applied in three independent biological experiments resulting in the quantification the EGFR and the six receptor-associated proteins (Fig. 5 and Table II). The EGFR peptide YSFGATCVK (residues 285–293; described above), which is common to the WT and variant proteins and not subject to phosphorylation, was quantified and used to normalize for the amount of EGFR in each sample.

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