Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

  1. Ryo Maeda
  2. Hiroko Tamagaki-Asahina
  3. Takeshi Sato
  4. Masataka Yanagawa
  5. Yasushi Sako

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Abstract

The cytoplasmic domain of receptor tyrosine kinases (RTKs) plays roles as a kinase and a protein scaffold; however, the allocation of these two functions is not fully understood. Here, we analyzed the assembly of the transmembrane (TM)–juxtamembrane (JM) region of EGFR, one of the best studied members of RTKs, by combining single-pair fluorescence resonance energy transfer (FRET) imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (T654) that is phosphorylated after ligand association. We observed that the TM–JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias toward oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of T654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which T654 phosphorylation causes a switch of the major function of EGFR from kinase-activating dimers to scaffolding oligomers.

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  1. Version published to 10.1242/jcs.260355
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    Referee #3

    Evidence, reproducibility and clarity

    The work of Sako and co-workers tries to employ a single (two) molecule technique in nanodisks to learn about EGFR TM-JM dimerization versus oligomerization. The authors vary the lipid composition in the disk as well as the phosphorylation state of Thr654 which resides in close proximity to several basic amino acids. The outcome suggested by the authors is that Thr654 phosphorylation switches EGFR from a signaling state to a scaffold state. This a tough problem to investigate and single molecule techniques such as FRET might be a successful way of finding answers. I should mention that I am not a single molecule specialist.

    The authors also performed live cell experiments in CHO cells that do not express endogenous EGFR. The results in figure 9 are interesting. However, receptor internalization has not been mentioned.

    In general, the work is premature for publication. My major critique points are 1) that there is a lack of statistical analysis in most of the figures and 2) that the lipid composition lacks highly negatively charged phosphoinositides, which are known to bind EGFR at the membrane interface. Likewise, what do we learn from artificial lipid compositions that lack cholesterol? Even non-phase-separated membrane areas contain cholesterol (maybe less). 3) Receptor internalization is not discussed.

    Major critique points:

    Figure 2. FRET changes after acceptor photobleaching. The excitation and emission wavelengths need to be stated. It is not clear from the traces that there is only a single donor peptide in the disk. Only the traces in the two left panels and the one in the upper right support FRET between Cy3 and Cy5. The "FRET efficiency" is misleading because it suggests that all traces show an increase in donor fluorescence after acceptor bleaching which is clearly not the case. Why is the donor fluorescence dropping? Donor bleaching? Figure 2a supports this. If so, this gives a nonsense FRET readout. There should be an accompanying bar graph with full statistical analysis. There should be control experiments in which two Cy3-tagged EGFR TM-JM molecules are used and photobleaching is still aiming for Cy5.

    Figure 3. There is no statistical analysis. As said for Figure 2: FRET efficiency seems only partially reflecting the single fluorophore traces. A change from 0.9 to 0.95 FRET efficiency seems small and statistical relevance is not determined.

    Figure 4. The result that cholesterol overwrote the Thr-P effect means that phosphorylation is not relevant in the cellular environment. Does this not contradict the main hypothesis?

    Figure 5. It is unclear how fluorescence intensity of CY3-labelled EGFR TM-JM peptides change. Is this due to homo-FRET? Again, a lack of cholesterol is not meaningful.

    Figure 9. I don't understand the results from the EGFR expression control. 30 min after EGF stimulation, the EGFR should have been internalized and destroyed in the lysosome. Why is there more EGFR? Do CHO cells lack the machinery to internalize RTKs? Again, no statistics in Figure 10.

    Significance

    As the test system is very artificial, I don't see major impact. The role of Ser/Thr phosphorylation sites is investigated quite substantially.

    I think the study should be re-designed. The cell work is quite interesting but major factors such as receptor internalization have not been considered.

    Audience: cell biologists

    My expertise: we work on EGFR signaling and EGFR phosphorylation.

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    Referee #2

    Evidence, reproducibility and clarity

    The main goal of this paper is to investigate the assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, using single-pair FRET imaging and a nanodisc technique. In particular, the authors address the role of a threonine residue (Thr654) that is phosphorylated after ligand association and is located in the JM domain of EGFR. The authors showed that anionic lipids, cholesterols, and EGFR Thr654 phosphorylation (pT654) regulate the dimerization and/or oligomerization of EGFR.

    Previously, these authors reported that anionic lipids caused the dimerization of JM domains, and that pT654 together with acidic lipids induced the dissociation of the EGFR dimer. In this manuscript, authors show that EGFR dimers assembled in cholesterols vs. anionic lipids display significant conformational changes mainly concerning the positioning of JM and TM. Furthermore, they state that phosphorylation of Thr654 (pT654) promoted oligomerization of TM-JM peptides in cholesterol containing membranes. Finally, the authors show distinct pT654-dependent functional roles and oligomerization states for EGFR in living cells. Authors propose a model in which membrane cholesterol and pT654 work cooperatively to switch the EGFR function from the kinase dimer to the scaffold oligomer

    Major comments:

    •Not clear what Figure 2 is supposed to indicate? Not clear what peptides are being tested. There is an indication in figure legend that photobleaching was performed but it is not clear how, when or why? If the idea is to validate FRET signal and analysis, then maybe photobleaching was performed to obliterate the acceptor dye and unquench the donor dye? If that is the case the authors must explain the experiment and what they are achieving with it. Maybe showing unquenching of the donor would be advised. Also, there is no mention in the text of the photobleaching experiment. If the goal of figure 2 is only to validate FRET analysis, then maybe it should be moved to suppl data.

    •Figure 3-4 address the role of cholesterol and pThr654 on the positioning of TM or JM. TM domains come closer in the presence of pT654 and membrane cholesterol. However, cholesterol but not pTh654 increased the proximity between the C-terminus of JM domains in PC 152 and PC/PS membranes. Although interesting results, the connection between the TM and JM conformational changes and oligomerization results is not direct or even interpretable into a model. The experiment that may be lacking is the testing of the role of cholesterol and pThr654 on the positioning of JM vs TM peptides. These experiments were suggested in Figure 2 but Figure 3-4 test the conformational changes of TM or JM but not of TM vs JM. Actually, Table 1 includes the values for Fig 3-4 FRET measurements which are TM-TM and JM-JM not TM vs JM as suggested in the title. The authors need to clarify this issue.

    •Figure 5: The authors suggest that a cooperative effect of cholesterol and Thr654 phosphorylation induces higher-order assembly of the TM-JM peptides. The relationship between these experiments and that of Figure 3-4 needs to be better described.

    • igure 6-7: Authors state that in all conditions other than non-phosphorylated peptides in the PC/PS membrane, Cy3 distributions after Cy5 photobleaching (red) peaked at the fluorescence intensity of ~100. However, the absence of statistical analysis of the comparison between different distributions makes this figure difficult to interpret and analyze. If not possible then the authors should stress that the results interpretation is based solely on a qualitative analysis of the curves.

    •Figure 8 model in which PS and cholesterol are proposed to exert competitive effects on dimerization and oligomerization, while pT654 disrupts the PS-induced JM dimer, and promotes oligomerization of the peptides, is not readily supported by the presented data.

    •Regarding the experiments of EGFR activation/phosphorylation using pT654 and pY1068 in Figure 9.

    -Blots should be clearly labeled (mainly pEGFR versus total EGFR and pT654 versus pY1068). Can authors clarify why in panel a only is showed 0 and 30 min (in pY1068 there is 3 time points) and why two main bands (one about 160 kDa and another about 210 kDa) are observed in all phospho or total EGFR immunoblots?

    -In panel a, it would be expected a stronger pT654 signal upon EGF in wt PMA(-)? Additionally, here the total EGFR blot is missing.

    -In panel b, it is important to clarify what band(s) are being quantified and statistical analyses (t-test or ANOVA for example) should be provided. Also, the graph indicates an about 2-fold increase in pY1068 signal at time 0 when wt PMA(-) is compared with pT654A PMA(-). This is apparently given by low total EGFR in pT654A condition, perhaps lower levels of transfection? it would be also a plus if it is possible to have beta actin or other loading control for blots or mention lower levels of transfection if it is the case.

    -Figure 9 presentation and analysis should be improved since it is an important part of main conclusions of the present work. Did authors considered to analyze also the effect a phosphomimetic in T654?

    •Figure 10b:

    -this reviewer cannot detect any difference between conditions/experiments, if there is something going on, authors need to find a better way to visualize the results and use statistical analysis to determine significance. Considering the lack of statistical analysis, Figure 10d overinterprets results since 10a-c do not provide strong supporting claims.

    -In methods authors mention that fluorescence is measured at the basal plasma membrane. However, it is not clear why at the condition pma- egf- the signal of egfr-gfp is so weak. Also, it not very clear why convexness indicates high immobilization.

    The following experiments and claims are over-interpreted or speculative. The reviewer would suggest limiting the conclusions extracted from these experiments. Moreover, Discussion is too long, convoluted, and speculative.

    •Oligomerization experiments are not convincing, and they are overinterpreted

    •homo-FRET (self-quenching) between two N-terminal labeled Cy3 peptides are speculative and not clear how to validate and demonstrate the presence of home-FRET. Maybe fluorescence-anisotropy-based homo-FRET detection could be included.

    Minor comments:

    •This sentence "Fractions of PS 97 (inner leaflet) and cholesterol (inner and outer leaflets) mimicked those in the plasma membrane" should be referenced

    •Figure 2a: the authors should explain what is the meaning of 5 seconds between the two images?

    •Figure 3 legend should include what type of peptide was used. According to the text the N-terminal regions of the TM domains were used in Fig. 3. However, that is not included in Fig 3 figure legend.

    •Figure 3-7 need the distribution and the 95% percentile section as in Figure 1 for statistical analysis of FRET measurements

    •Figures 9 and 10 should include statistical analysis to demonstrate significance

    Significance

    It should be mentioned that a significant amount of work included in this manuscript was already extensively covered in previous publications of the group. In particular, this paper follows the previous one "Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor" (Biophys J. 2018 Feb 27; 114(4): 893-903), where authors evaluate the effects of anionic lipids on EGFR dimerization. The authors cite this previous work in the manuscript, but nevertheless the first figures of the present manuscript are very similar to the previous work mentioned. The only difference being that the authors now are adding cholesterol as a new variable. Moreover, text throughout the main part of the manuscript (especially description of Results and Introduction) are very similar to the previous work published. Overall, this similarity between results and approach, decrease the significance and novelty of the present work.

    Maeda et al., describes an advance regarding the role of cholesterol in EGFR molecular assembly. This is compared here to the previous findings about effect of phosphorylation in EGFR Thr654 residue and anionic lipids in EGFR molecular assembly. Since EGFR is a widely studied receptor both in health and disease, especially in cancer, the mechanisms of EGFR activation and assembly are of high interest both in basic and clinical research. It would be interesting if the authors of the present work could contextualize the physiological relevance of the findings and how for instance depletion or increase in cholesterol physiologically could impact in EGFR related functions.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript titled "Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids", Maeda et al. investigate the associations of the EGF receptor, in particular the short TM-JM linker peptide that connects the extracellular to the intracellular domain and couples ligand binding to kinase activity. A particular focus lies on the influence of cholesterol and the phosphorylation of T654 on this process. Based on single-molecule FRET and intensity measurements in lipid nanodiscs, biochemical activity assays and analysis of EGFR cluster formation in live cell, they conclude that phosphorylation of T654 promotes clustering through dissociation of anti-parallel JM dimers and cholesterol supports the clustering process. In particular, they challenge the view that clusters contain kinase-active EGFR dimers, but rather are tight assemblies of EGFR in form with reduces kinase activity. This is a very interesting story and could explain observations made by the authors and others, e.g. decreased activity of EGFR after initial clustering. The experiments are convincing, but the authors should revisit the interpretation of their results; in particular, they should consider the possibilities (i) that nanodiscs might have different sizes depending on the lipid composition, (ii) that a bias towards repeated interactions is introduced when selecting nanodiscs containing three peptide molecules. Also, the normalization to basal levels of EGFR expression and activity in the Western blot and luminescence assay should be treated more carefully. Nevertheless, it is a good story that would be a good contribution to the EGFR field.

    Major comments:

    1.Line 93: the authors write that with cholesterol, there were two peaks. But there are three peaks (Fig. 1e, red line). I understand the following sentence that they used the peak at 7-8mL for their experiments and the analysis in Fig. 1f. They then write that this fraction had a similar size without cholesterol. But I see only one peak without cholesterol (at 11-13mL), and it shows a bigger size, as they also wrote before. Or do they refer to the very small bump in the black line at 7-8mL?

    2.Figure 6: If the density of proteins in the lipid is low (see previous comment), the selection of nanodiscs with two Cy3 and one Cy5 introduces a bias, in particular in the conditions where there is no higher-order assembly occurring (i.e. with non-phosphorylated T654). Since the three proteins are then restricted to a very small membrane area, they are experiencing a high apparent density, and the equilibrium between assembled and dissociated 3-protein clusters will be shifted to the trimer.

    3.Figure 6: In connection to the previous comment, the previous comment #1 becomes very critical. If I understand the red line (PC/PS+chol) and black line (PC/PS) of Fig. 1 correctly, the size of the nanodiscs is bigger for PC/PS than for PC/PS+cholesterol. If this is the case, it will have an impact on the apparent density of the three protein molecules in the nanodisc, since a larger nanodisc means lower membrane density or lower encounter probability. Also, a quick MC simulation shows that the average distance of two randomly positioned proteins in a disc is 0.9*radius, which would be 5nm for the 11nm nanodiscs, and therefore within the Forster radius of Cy3 and Cy5 (5.3nm). With larger nanodiscs, this would change and less FRET would occur in the dissociated state. Therefore, the size of the PC only and PC/PS nanodiscs should be measured similar to the PC/PS+cholesterol. The effects of nanodisc size on apparent density and on FRET in the dissociated state should be considered.

    4.Fig. 9b: It seems that the T654A mutation increases EGFR expression since the EGFR-normalized levels are higher in wt, but lower in T654A. Was this effect directly quantified, e.g. by comparing to a standard protein? How can this be explained? The authors claim that pY1068 levels were higher for T654A than for WT. Although this is correct, the levels normalized to total EGFR were lower for T654A than for WT. This is somehow contradictory and should be resolved.

    5.Is it possible that basal Grb2 recruitment is different for WT and T654A, and therefore the baseline for the luminescence measurement is different? This should be carefully investigated, since Fig. 9b suggests that T654A has a critical impact on EGFR expression in its inactive state, and therefore can be assumed to influence basal EGFR activity.

    6.Line 401: The claim that pT654 promotes downstream signal transduction is based on the apparently increased EGF-induced Grb2 recruitment in the presence of PMA (which leads to T654 phosphorylation) in the EGFR wt but not in the T654A mutant. This claim might not hold when considering that maybe basal levels of Grb2 interaction were different under the different conditions. Then the apparent increase would only be caused by the normalization to the basal luminescence level (see comment #5).

    Minor comments:

    1.Line 66: the first work should probably be "although" instead "also".

    2.Line 101: it is unclear what "16 types of nanodiscs" mean. Were there more lipid compositions than the four compositions in Fig. 1d investigated? Or was it always nanodiscs prepared with the same lipid composition (and same fraction) but 16 different proteins or combinations of proteins?

    3.Fig. 3-7 (all FRET and intensity distribution diagrams): An error for a each bin should be calculated or estimated so it is easier to assess if a bump in the line is more than random, and if the difference between two distributions is significant.

    4.Line 128: "PS competed with cholesterol when T654 was non-phosphorylated (Fig. 3d)." What does compete exactly mean here and how is this deduced from the data?

    5.Line 139: "These results confirmed the results of our previous study". The authors could clarify this by stating what the conclusion was/is (T654 phosphorylation dissociates dimers when acidic lipids are present).

    6.Figure 5: adding the conditions into the panels would clarify the figure.

    7.Line 167: In how far can be assumed that co-localization of multiple peptides in the same nanodisc is caused by interaction? Could they not be incorporated by coincidence, because of a high density? What is the probability to obtain two non-interacting proteins in the same nanodisc?

    8.Figure 6: I cannot find the details to determine pre- and post-bleaching intensities. The authors should state in the methods the time they include to determine the average value on each side of the bleaching event. The same for all values where intensities or FRET efficiencies were calculated by averaging over multiple points of the time course.

    9.Line 177: If the low intensity for N-terminally labeled peptides was caused by homo-FRET, there should be an increase of intensity after the first Cy3 bleached. Did the authors observe that? Also, when the trajectories are getting complicated by homo-FRET during dissociation/association, the selection of nanodiscs with two Cy3 might become ambiguous. What were the exact selection criteria? There is no information in the text or methods.

    10.Figures 6a/7a: The authors should double check the origin of the data. The panels in the bottom left of Fig. 6a and the bottom left of Fig. 7a seem to contain identical data.

    11.Figure 7b-e: Although there are differences in the intensity histograms, I find them to be quite subtle, compared to the effect of pT654. Therefore, I find the claims made in lines 224-229 (induction of oligomerization by cholesterol, and oligomerization instead of dimer by pT654) too strong. Based only on the visual assessment of the intensity distributions, I would rather suggest a shift towards one or the other condition. A model calculation with assumptions for FRET values and distances for certain conditions would strengthen their claims.

    12.Figure 8: The different configurations and why they are induced in certain condition needs to better explained. In the main text, there is only one sentence (lines 224-229), and in the figure legend, there is no explanation at all.

    13.Line 230: "...whether the peptides in the oligomers directly interacted or not." Since there are no other proteins involved except the peptides, I am wondering how an indirect interaction in the oligomer would work?

    14.Fig. 10b: It is surprising that the authors observe a large fraction of oligomers for EGFR WT without EGF or PMA, since EGFR WT is thought to be primarily monomeric in its inactive state. This should be discussed.

    15.Fig. 10b: The distributions might better reflect the process of oligomerization if the fraction is multiplied by the size of the oligomer. This would show reflect how many receptors are clustered, instead of how many clusters are present (one large cluster should make the same fraction as the sum of two smaller clusters half the size).

    16.Fig. 10a: The authors should present high quality movies of the observations to allow the reader to appreciate the clustering/immobilization better and also to facilitate the comparison of raw data for other researchers in the field.

    17.Many experiments in this work assume that the intensity of a fluorophore remains constant while it remains in a specific monomer or dimer state. This should be confirmed with nanodiscs containing positive and negative control proteins for dimerization. E.g. a monomeric protein should give a constant intensity, and a constitutively dimeric protein should give two intensity levels, one before and one after photobleaching of the first dye molecule. Also, FRET should remain quite constant over time in a constitutive dimer labeled with Cy3 and Cy5.

    18.It is questionable in how far the postulated trimer formation will happen if the extracellular and kinase domains are present in the full EGFR, since these domains are quite bulky and might not allow certain trimer configurations, e.g. the close trimer of the JM domain. This should be discussed.

    Significance

    The prevalent view that EGFR clusters contain kinase-active dimers is challenged. The authors claim that instead, tight assemblies of EGFR in form with reduces kinase activity after the initial activation phase. Based on the novel lipid nanodisc FRET experiments, different configurations of the TM-JM domain can be discriminated in the membrane environment. This study is a good contribution to the EGFR field and will be interesting also for people in the single-molecule imaging field. The reviewer has experience in single-molecule imaging and has also worked on the EGFR.

    Referess cross-commenting

    I want to add two comments:

    1. For reviewer #2 's first major comment on the photobleaching: From my point of view as a single-molecule researcher, this experiment is quite clear. Photobleaching is a process that inevitably occurs when using high intensity, as required for single-molecule imaging; therefore, it was not "performed", but (unfortunately) occurs. Only the data before the photobleaching event are useful. But I agree with the reviewer that Fig. 2 could be moved to the supplement, or, in a condensed form, could be merged, e.g. with Fig. 3 and Fig. 4.

    2. Concerning reviewer #2 's significance statement: I was not aware of the fact that the nanodisc approach has been used and published by the same authors before. They should have clearly indicated this in a written form, e.g. by saying that they used a previously developed nanodisc FRET assay. Also there is no reference to the previous work in the first results section or the nanodisc section of the methods. This diminishes the significance and novelty of the work considerably in my opinion. Also, as the reviewer correctly observes, in the text of this manuscript there are a number of similarities to the mentioned publication which also reduces the originality of this work.

  6. Version published to 10.1101/2021.05.14.444132v4 on bioRxiv
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