Clustering-based positive feedback between a kinase and its substrate enables effective T-cell receptor signaling

Protein clusters and condensates are pervasive in mammalian signaling. Yet how the signaling capacity of higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. Here, we present an optogenetic approach to switch between light-induced clusters and simple protein heterodimers with a single point mutation. We apply this system to study how clustering affects signaling from the kinase Zap70 and its substrate LAT, proteins that normally form membrane-localized clusters during T cell activation. We find that light-induced clusters of LAT and Zap70 trigger potent activation of downstream signaling pathways even in non-T cells, whereas one-to-one dimers do not. We provide evidence that clusters harbor a local positive feedback loop between three components: Zap70, LAT, and Src-family kinases that bind to phosphorylated LAT and further activate Zap70. Overall, our study provides evidence for a specific role of protein condensates in cell signaling, and identifies a simple biochemical circuit that can robustly sense protein oligomerization state. Highlights- A general system for studying the role of protein clusters versus dimers. - Membrane clusters of the kinase Zap70 and its substrate LAT trigger potent downstream signaling. - Clustering Zap70 with LAT is required for full activation of Zap70 kinase activity. - A positive feedback loop connects phosphorylated LAT to Zap70 activation via Src-family kinases.