Beyond signaling activation: Phosphorylation modulates Grb2 phase separation to create multivalent scaffolds

The Growth Factor Receptor-Bound Protein 2 (Grb2) is a central adaptor protein in signal transduction pathways, yet how its monomer-dimer equilibrium governs its supramolecular organization remains elusive. Here, we demonstrate that the oligomeric state of Grb2 acts as a binary switch for Liquid-Liquid Phase Separation (LLPS). While the auto-inhibited homodimer forms only transient, thermodynamically unstable assemblies in crowding conditions, the phosphorylation-mimetic monomer (Y160E) drives the formation of robust, gel-like condensates. Integrating turbidity assays, temperature-controlled dynamic light scattering, fluorescence recovery after photobleaching (FRAP), hyperspectral imaging analyses, and coarse-grained molecular dynamics simulations, we reveal that this phase transition is enthalpy-driven and reliant on a specific electrostatic network between the SH2 domain residue R142 and a C-terminal SH3 acidic cluster (Q170-D172). Crucially, we show that these monomeric condensates function as "scaffolds" that efficiently recruit and sequester cytosolic wild-type dimers ("clients") into the dense phase. This recruitment mechanism resolves the paradox of how non-condensing wild-type proteins participate in phase separation. Our findings propose a novel regulatory model where phosphorylation nucleates the formation of high-density signaling hubs, redefining Grb2 from a passive adaptor to a dynamic spatial organizer of the Ras/MAPK pathway.