Chun-Wei Lin
Chun-Wei Stamp


A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane

Chun-Wei Lin, Laura M. Nocka, Brittany L. Stinger, Joseph B. DeGrandchamp, L.J. Nugent Lew, Steven Alvarez, Henry T. Phan, Yasushi Kondo, John Kuriyan and Jay T. Groves

Proc. Acad. Sci. USA 2022 119(19):e2122531119. doi: 10.1073/pnas.2122531119     (local copy)
BioRχiv 2021.12.12.472247; doi: https://doi.org/10.1101/2021.12.12.472247

Abstract

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ~200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Figures from the paper

(Click on the small image to get a higher-resolution version.)
Figure 1 from paper

Figure 1 - EGFRTAIL phase transition induced by the dimerization of Grb2.


(A) The schematic of the reconstitution experiment. The phosphorylated EGFRTAIL is tethered on the supported membrane. EGFRTAIL has several tyrosine sites that can be phosphorylated including Tyr 1068, Tyr 1086, Tyr 1148 and Tyr 1173 related to Grb2 recruitment. Grb2 is injected to the chamber and recruited by EGFRTAIL. Grb2 is shown to dimerize on the supported membrane. The dimerization of Grb2 drives the assembly of EGFRTAIL:Grb2 into phase transition. EGFRTAIL is initially in the state where it is mobile on the supported bilayer (left). EGFRTAIL becomes part of the protein condensate after the phase transition is triggered (right).
(B) TIRF images of Alexa Fluor 488 labeled EGFRTAIL on the supported bilayer revealing a phase transition within 2 minutes after the addition of Grb2 to form a condensed phase of 950 molecules/µm2 density. The protein condensate of EGFRTAIL:Grb2 continues to mature over 10 minutes to achieve densities of ~3760 molecules/µm2. The bottom figures are zoomed-in images of the lower-left corner in the upper images. The inserted figures at the upper right corner are the 2D Fourier Transform of the TIRF images, which confirm that the structure of the condensate is isotropic.
Figure 1 from paper

Figure 1 continued- EGFRTAIL phase transition induced by the dimerization of Grb2.


(C) The macroscopic fractional area coverage of the EGFRTAIL:Grb2 condensate, as well as its morphological features, scale with the density of EGFRTAIL. Image data here, spanning from 50 to 3000 molecules/µm2, illustrate EGFRTAIL:Grb2 condensate variation from small clusters (left) to large interconnected domains (right).
(D) The crystal structure of Grb2 shows the asymmetric dimer (PDB: 1GRI). The tyrosine 160 at the dimeric interface is illustrated in the zoom-in picture.
Figure 2 from paper

Figure 2 - The colocalization between EGFRTAIL and Grb2 after EGFRTAIL:Grb2 phase transition is achieved.


Unlabeled Grb2 mixed with Alexa Fluor 647 labeled Grb2 (0.2 µM, labeling efficiency of 63%) was added to phosphorylated Alexa Fluor 488 labeled EGFRTAIL to induce the phase transition of EGFRTAIL:Grb2. The TIRF images were taken 10 minutes after the addition of Grb2. The first image in green is from EGFRTAIL. The second image in magenta is from Grb2. The third image is the merged image from EGFRTAIL and Grb2.
Figure 3 from paper

Figure 3 - EGFRTAIL:Grb2 phase transition is reversed by phosphatase.


The first image is the TIRF image of EGFRTAIL condensation taken at 15 minutes after the addition of Grb2 to phosphorylated EGFRTAIL on the supported bilayer. The second image was taken after the addition of 1 µM phosphatase, YopH. EGFRTAIL:Grb2 condensate is rapidly dissolved in 1 minute. The last image taken at 3 minutes after the addition of YopH shows uniformly distributed EGFRTAIL (also see Figure S1).
Figure 4 from paper

Figure 4 - Grb2 dimerization as the key driving force to induce phase transition.


TIRF images of EGFR showing the reconstitutions of
(A) wildtype Grb2 and EGFRTAIL. Phase transition is observed at 3 minutes.
(B) Grb2Y160E and EGFRTAIL. EGFRTAIL remains uniformly distributed. Phase transition is not detected.
(C) pGrb2 and EGFRTAIL. Phase transition is not detected.
(D) wildtype Grb2 and EGFRTAIL without ATP. Phase transition is not detected.
(E) Grb2R86K, SH2 impaired Grb2 mutatnt, and EGFRTAIL. No phase transition is observed.
(F) wildtype Grb2 and LAT. Phase transition of LAT:Grb2 is observed in 6 minutes.
In (A-F), Hck is the kinase used to phosphorylate EGFRTAIL.

Figure 5 from paper

Figure 5 - The schematic of downstream signaling modulated by SOSPR titrated EGFRTAIL:Grb2 phase transition.


SOSPR was used as the strong crosslinker to induce the phase transition of the molecule assembly, EGFRTAIL:Grb2:SOS, at different levels. To initiate the downstream signaling, Grb2, SOSPR, GTP (1 mM), Alexa Fluor 555 labeled SOSFL and Alexa Fluor 647 labeled RBD (50 nM) were added together to the bilayer. Once SOSFL was activated, the activated SOSFL would stay on the membrane and processively activate Ras. The activated Ras (Ras-GTP) is detected by RBD. The downstream signaling at the activation of Ras is read by the fluorescence intensity of RBD on the bilayer. Also see Ras activation in Materials and Methods.
Figure 6 from paper

Figure 6 - Ras activation modulated by EGFRTAIL:Grb2 phase transition.


(A) The kinetic traces of Ras activation and the relative nucleotide exchange as the function at [SOS] = 0.2 nM.
(B) The kinetic traces of Ras activation and the relative nucleotide exchange as the function of time at [SOS] = 2 nM.
(C) The plot of the relative nucleotide exchange rate against the activated Ras showing the positive feedback of SOS at [SOS] = 0.2 nM.
(D) The plot of the relative nucleotide exchange rate against the activated Ras showing the positive feedback of SOS at [SOS] = 2 nM.


Supplemental figures from the paper

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Supplemental Figure 1 from paper

Supplemental Figure 1 - FRAP experiments of EGFR.


(A) FRAP images. Top: EGFRTAIL on the bilayer, middle: EGFRTAIL after YopH treatment, bottom: EGFRTAIL:Grb2 protein condensate.
(B) Intensity profile of FRAP image from EGFRTAIL only at 0 and 3 min.
(C) The recovery trace of EGFRTAIL only. The red solid line is the exponential fit where τD = 73 s.
Supplemental Figure 2 from paper

Supplemental Figure 2 - Grb2Y160E responses to the phosphorylation of EGFRTAIL.


Alexa Fluor 647 labeled Grb2Y160E (6 µM, labeling efficiency of 48%) is added to phosphorylated EGFRTAIL on the bilayer. The averaged fluorescence intensity from the TIRF images of Grb2Y160E on the supported bilayer increases rapidly corresponding to the recruitment by EGFRTAIL (the red curve in the left graph). The control experiment is shown by the black curve where EGFRTAIL is not phosphorylated. The TIRF images from Grb2Y160E (in magenta) and EGFRTAIL (in gray) taken 10 minutes after the addition are shown at the right side of the figure. No phase transition is observed using Grb2Y160E.

Supplemental Figure 3 from paper

Supplemental Figure 3 - Ras activation between wildtype Grb2 and Grb2Y160E.


In this Ras activation assay, the crosslinker, SOSPR is not used. To initiate the downstream signaling, Grb2 (200 nM), GTP (1 mM), Alexa Fluor 555 labeled SOSFL (4 nM) and Alexa Fluor 647 labeled RBD (50 nM) are added together to phosphorylated EGFRTAIL on the supported bilayer. The activated Ras (Ras-GTP) is detected by RBD. Also see Ras activation in Materials and Methods.

Supplemental Figure 4 from paper

Supplemental Figure 4


TIRF images of Alexa Fluor 488 labeled EGFRTAIL on the supported bilayer 10 minutes after the addition of Grb2 and SOSPR showing small amounts of SOSPR, such as were used in our detailed studies of the effect of EGFR condensation on SOS activity, are insufficient to drive condensation if the Grb2Y160E mutant is used—thus a Grb2:Grb2 interaction appears to be essential, at least under those conditions. At high enough SOSPR levels, it is possible to overcome the need for the Grb2:Grb2 interaction in reconstituted experiments.