Abstract
The ∼230 residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail, and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and find that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80 residue span is important for autoinhibition, and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.
Figures from the paper
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Figure 1 - Model for activation of epidermal growth factor receptor (EGFR) and constructs used in this study
A. Ligand binding to the extracellular domain of the epidermal growth factor (EGF) receptor induces a conformational change that results in receptor-mediated dimerization and activation. Activation of the intracellular kinase domains is promoted by the formation of an asymmetric dimer, in which one kinase domain (the activator; yellow) activates the other (the receiver; blue).
B. Domain architecture of human EGFR with domain boundaries highlighted. The domain composition of the EGFR-family constructs used in this study is also presented, including EGFR deletion constructs, the EGFR-HER3 tail chimera and HER2 (Δ - deletion; mCh – mCherry fluorescent protein fusion).
Figure 2 - Effect of deletions in the EGFR tail on EGFR phosphorylation
A. The vIVb deletion in the proximal region of the EGFR tail enhances autophosphorylation on Tyr 1068 and 1173, even in the absence of EGF stimulation. The upper panels represent the whole dataset. Data for wild-type and deletion mutants are shown as open and closed circles, respectively. Phosphorylation levels with or without EGF stimulation are show in red and blue, respectively.
B. Same as in A, but showing data for cells expressing the ∆(999-1186) EGFR mutant with a deletion in the distal region of tail. The levels of phosphorylation detected for Tyr 974 and Tyr 992 are substantially lower than those for the wild-type.
C. The effect of the same deletion constructs as in A and B on the activation loop tyrosine (Tyr 845). The vIVb deletion in the proximal region leads to enhanced phosphorylation, whereas deletion of the distal region of the tail is necessary for activation loop tyrosine phosphorylation.
Figure 3 - An autoinhibitory function of the EGFR C-terminal tail maps to the entire proximal region deleted in the vIVb mutant
A. Illustration of overlapping deletions mutants scanning the EGFR C-terminal tail.
B. Flow cytometry analysis of Tyr 1173 phosphorylation with EGFR tail mutants, with and without EGF stimulation. The analysis was performed as described in Figure 2, and phosphorylation levels are normalized to unstimulated, wild-type EGFR-expressing cells.
C. Flow-cytometry analysis of phospho-Erk1/2 (pErk) in cells expressing the vIVb deletion mutant (∆(vIVb)) or selected deletion mutants depicted in A. Histograms of pErk signal are shown for cells expressing moderate amounts of EGFR constructs, with or without EGF stimulation. The data for each mutant are separately plotted, overlaid on the data for wild-type EGFR.
Figure 4 - The vIVb deletion increases EGFR phosphorylation only when present in both the activator and receiver of the active asymmetric dimer
A. Flow cytometry analysis of activation loop phosphorylation using co-transfected EGFR mutants. Pairs of EGFR constructs consisting of activator-impaired, mCherry-tagged EGFR, and receiver-impaired, Cerulean-tagged EGFR were co-transfected into HEK-293T cells. After EGF stimulation, cells were analyzed for mCherry, Cerulean, and FITC fluorescence, reflecting the expression levels of each construct and anti-phosphotyrosine staining for Tyr 845.
B. Flow cytometry analysis of phospho-Erk1/2 (pErk) in cells expressing mutant EGFR asymmetric dimer pairs. Cells containing the pairs of constructs described in A were treated with or without EGF and stained for pErk. Each ∆(vIVb) combination (deletion in the activator-impaired construct, receiver-impaired construct, or both) is plotted separately, and overlaid on with the intact-tail pair (wild-type).
Figure 5 - The NPXY motif encompassing Tyr 1086 of EGFR is required for Tyr 845 phosphorylation in HEK-293T cells.
A. Flow cytometry analysis of Tyr 845 phosphorylation with EGFR tail deletion mutants, with and without EGF stimulation.
B. Flow cytometry analysis of Tyr 845 phosphorylation with EGFR mutated either at Tyr 1086 or with the deletion of residues 1083–1086 (∆NPXY).
C. Western blot analysis of Tyr 845 phosphorylation with and without EGF stimulation for Y1086A and ∆NPXY mutants.
D. Flow cytometry analysis of Tyr 974 and Tyr 992 phosphorylation for Y1086A and ∆NPXY mutants.
E. Flow cytometry analysis of Tyr 845 phosphorylation with fine-scale scanning deletion mutants.
Figure 6 - Crystal structures of EGFR V924R and I682Q mutants
A. Superposition of the EGFR I682Q structure here presented (light blue) with the EGFR structure bound to Mig6 (white; PDB code 2RFE; chain A). The electron density at the base of the C-lobe of monomer A in the EGFR I682Q structure is shown, for a map calculated with the coefficients 2mFo-DFc (purple) and mFo-DFc (green positive peaks) and phases obtained from a model at the late stages of refinement.
B. Orthogonal zoomed views of the region delimited in A by a rectangle.
Figure 7 - Proposed model for activator interface occlusion by the C-terminal tail
A. Molecular dynamics simulation snapshot ~5 ps after spontaneous formation of an α-helix including Phe 999 and Phe 1000 of the tail (FF helix).
B. Overlaid snapshots at 7 ps and 307 ps after formation of the FF helix.
C. Comparison of the conformation of Mig6 and the EGFR tail model after simulation.
Figure 8 - Tail phosphorylation in the activator and receiver kinases
A. and B. Flow cytometry analysis of Tyr 1173 phosphorylation by co-transfected EGFR mutants. mCherry (EGFR–mCh) and Cerulean-tagged (EGFR–Cer) versions of EGFR were co-transfected into Cos7 cells, and were analyzed for mCherry, Cerulean, and FITC fluorescence, reflecting the expression levels of each construct and anti-phosphotyrosine staining for pTyr 1173 after EGF stimulation.
C. Mean phosphorylation level plotted against expression level for bins on the diagonal in Bs, reflecting cells expressing similar levels of mCherry- and mCerulean-tagged EGFR (±SEM). Phosphorylation at Tyr 1173 increases with expression level significantly more when the EGFR tail is on the activator compared to when it is on the receiver.
Figure 9 - An EGFR-Her3 tail chimera produces higher phosphorylation levels when it takes the activator position in an asymmetric dimer
A. Schematic illustrating the combinations of constructs described in B. and C.
B. Phosphorylation of Her3 Y1289 analyzed by flow-cytometry.
C. Relative phosphotyrosine signal for each pair of constructs for intermediate expression levels (500–600 mCherry fluorescence units as shown in B), normalized to the unstimulated EGFR -Her3 tail signal (error bars, ±SEM).
Figure 10 - C-terminal tail phosphorylation by EGFR/Her2 heterodimers is greater when EGFR takes the receiver position
A. Schematic illustrating the combinations of constructs described in B. and C.
B. Phosphorylation of Her2 Tyr 1221 and EGFR Tyr 1173 upon EGF stimulation analyzed by flow-cytometry.
C. Phosphotyrosine signal for each pair of constructs for intermediate expression levels (500–600 mCherry fluorescence units), as shown in B (error bars, ±SEM).
Figure 11 - Vesicle-reconstituted TM-ICM EGFR exhibits biphasic autophosphorylation kinetics
A. Time courses of EGFR autophosphorylation by TM-ICM EGFR reconstituted into vesicles determined by Western blotting with the indicated antibodies.
B. Time courses of phosphorylation reactions as quantified by tandem mass spectrometry.
Figure 12 - A short-tailed EGFR TM-ICM construct incorporated into vesicles does not exhibit biphasic autophosphorylation kinetics
A. Negative stain electron micrograph of vesicles reconstituted with EGFR TM-ICM truncated after residue 998 (left), and illustration of the location of phosphorylated tyrosines in this construct (right).
B. Time courses of autophoshorylation on the indicated tyrosines for the short-tail TM-ICM incorporated into vesicles, as quantified by Western blot.
Supplemental figures from the paper
(Click on the small image to get a higher-resolution version.)
A. Empty vesicles. Enlarged image of panel in Fig. 11A.
B. EGFR TM-ICM reconstituted in vesicles leads to membrane stacking. Enlarged image of panel in Fig. 11A.
Supplemental Figure 1 - EGFR sequence alignment
Supplemental Figure 2 - Analysis of EGFR phosphorylated tyrosine antibody specificity
Supplemental Figure 3A - Full flow cytometry data for the EGFR tail deletions presented in Fig. 3B - EGFR tail phosphorylation (Tyr 1173)
Supplemental Figure 3B - Full flow cytometry data for the EGFR tail deletions presented in Fig. 3B - EGFR activation loop phosphorylation (Tyr 845)
Supplemental Figure 4 - Data analysis of the flow cytometry essay measuring pErk accumulation as a function of EGFR expression and activation
Supplemental Figure 5 - Crystal structures of EGFR V924R and I682Q mutants
Supplemental Figure 6A - Molecular dynamics simulations - α-Helix formation over time
Supplemental Figure 6B - Molecular dynamics simulations - β-sheet Hydrogen Bond Distances for the Mig6-like tail segment
Supplemental Figure 7 - Roles of the LLSSL (residues 1010-1014) segment in autoinhibition
Supplemental Figure 8 - HER3 tail phosphorylated tyrosine antibody specificity
Supplemental Figure 9 - Vesicle-reconstituted TM-ICM EGFR exhibits the same biphasic autophosphorylation kinetics after phosphatase treatment
Supplemental Figure 10 - Incorporation of a TM-ICM construct of EGFR into unilamellar vesicles induces membrane stacking
Supplemental Figure 11 - Electron cryomicroscopy images
A. Empty vesicles. Enlarged image of panel in Fig. 11A.
B. EGFR TM-ICM reconstituted in vesicles leads to membrane stacking. Enlarged image of panel in Fig. 11A.
