Lew, L. J. N. | Search bioRxiv

bioRxiv2026

Supported membrane assay probes PLCγ1 activity in LAT condensates

Phospholipase C-{gamma}1 (PLC{gamma}1) plays a critical role linking T cell receptor activation with downstream signaling pathways including calcium. PLC{gamma}1 activation in T Cells relies on phosphotyrosine-mediated recruitment to the membrane-bound scaffold LAT, which becomes crosslinked through a bond percolation network with Grb2 and other scaffold and signaling molecules to form a signaling condensate. PLC{gamma}1 in these LAT condensates becomes activated, leading to induction of extracellular calcium influx. While PLC{gamma}1-driven calcium signaling is clearly correlated with LAT condensation, it is less clear how--or if-- the LAT condensation state facilitates PLC{gamma}1 activity. Here we develop an image-based PLC{gamma}1 activity assay in supported bilayers that enables simultaneous measurement of both PLC{gamma}1 recruitment to phosphorylated LAT and PLC{gamma}1-catalyzed hydrolysis of PIP2 in the membrane. The condensation state of LAT is independently controlled by adjusting levels of co-condensation proteins such as Grb2, SOS, GADS, and SLP76. The hydrolysis product, diacylglycerol (DAG), remains in the membrane and is monitored as a readout of catalytic activity using a DAG sensor based on the C1b (DAG binding) domain of PKC{theta}. Assays are performed directly with mammalian cell lysate containing fluorescent PLC{gamma}1 fusion constructs. The results reveal that PLC{gamma}1 is highly active when recruited to dispersed LAT and that the condensed state does not promote activity. Overall, this assay platform reveals that despite the correlation between PLC{gamma}1 signal gating and LAT condensation, the physical environment of the condensate itself is not a key regulator of PLC{gamma}1 signaling. More broadly, this assay system offers a quantitative means of probing how PLC{gamma}1 activity is controlled at the membrane.

biophysics↗

bioRxiv2024

Grb2 Phosphorylation Antagonizes EGFR-driven Ras Activation by Interfering with Condensate Assembly

Upon ligand binding, the kinase domain of EGFR phosphorylates multiple tyrosine residues on the receptor cytoplasmic tail through a trans-autophosphorylation process. Phosphotyrosine sites on activated receptors recruit Grb2, which further recruits SOS to initiate downstream signaling by activating Ras. Multivalent binding between SOS and Grb2, as well as direct Grb2:Grb2 interactions, contribute to formation of a protein condensate of activated EGFR. The condensed state of EGFR facilitates autoinhibition release in SOS and exerts regulatory control over signal propagation from activated EGFR to Ras. While kinase activity of EGFR is an essential driver of this signaling process, phosphorylation at residue Y160 on Grb2 blocks Grb2:Grb2 binding and can interfere with EGFR condensation. Here, using a reconstituted system, we examine how titrating kinase activity in the EGFR system can both promote and inhibit signal output to Ras. The results reveal how effects of tyrosine kinase inhibition can, under some circumstances, promote Ras activation by inhibiting negative feedback through Grb2 phosphorylation and disruption of a Grb2 SH2/SH3 dimer interface. Statement of SignificanceActivated EGFR forms a biomolecular condensate, via linkage of multiple EGFR through Grb2 and SOS, and the condensation state of EGFR influences signal propagation to Ras. While tyrosine phosphorylation is a critical step in EGFR activation, phosphorylation of Grb2 can have an inhibitory effect on EGFR condensation and subsequent Ras activation. Under some conditions, kinase inhibition can promote signaling from EGFR to Ras.

biophysics↗