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

Abstract

Significance Two-dimensional condensates of proteins on the membrane surface, driven by tyrosine phosphorylation, are beginning to emerge as important players in signal transduction. This work describes discovery of a protein condensation phase transition of EGFR and Grb2 on membrane surfaces, which is poised to have a significant impact on how we understand EGFR signaling and misregulation in disease. EGFR condensation is mediated through a Grb2-Grb2 crosslinking element, which itself is regulatable through a specific phosphotyrosine site on Grb2. Furthermore, the EGFR condensate exerts significant control over the ability of SOS to activate Ras, thus implicating the EGFR condensate as a regulator of signal propagation from EGFR to Ras and the MAPK pathway.

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

“…We have demonstrated recently that the R86K mutation impairs the ability of Grb2 to promote phase separation of scaffold proteins (C.‑W. Lin et al, 2022). Another Grb2 variant (Y160E) has a reduced capacity for dimerization (Ahmed et al, 2015).…”

“…Grb2 is responsible for bringing signaling enzymes to their substrates and also helps in the formation of signaling clusters at the plasma membrane by virtue of its ability to crosslink scaffold proteins or receptors (Huang et al, 2019; Huang, Chiang, & Groves, 2017; Huang et al, 2016; C.‑W. Lin et al, 2022; Su et al, 2016). For example, in the MAPK pathway, the SH3 domains of two Grb2 molecules bind to the Ras activator Son of Sevenless (SOS).…”

Protein conformation and biomolecular condensates

“…In turn, ZAP70 phosphorylates multiple tyrosine residues of the transmembrane protein linker for activation of T cells (LAT), which engages the SH2 and SH3 domain-containing protein GRB2 and SOS1 for activation of mitogen-activated protein kinase (MAPK) signaling. Phosphorylated LAT elicits phase separation with GRB2 and SOS1 ( Su et al., 2016 ; Lin et al., 2022 ).…”

Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes

“…SOS is capable of binding more than one Grb2 in this way and thus provides a cross-linking mechanism that leads to networked assembly of a LAT:Grb2:SOS protein condensate on the membrane surface ( 5 , 7 ). Other cross-linking interactions, such as a direct Grb2:Grb2 binding interface ( 18 ), may participate, but Grb2-mediated cross-linking is primarily limited to three LAT tyrosine sites, thus only achieving the minimum valency required for bond percolation. Another tyrosine site on LAT, which exhibits differential phosphorylation kinetics ( 19 ) and selectivity for the key signaling molecule phospholipase C-γ (PLC-γ) ( 20 ), provides a fourth cross-linking pathway that may be physiologically important in nucleation of the LAT condensate ( 12 ).…”