Phase-separated RTK fusion condensates orchestrate oncogenic signaling through extensive protein recruitment and RTK crosstalk

Liquid-liquid phase-separated RTK (Receptor Tyrosine Kinase) fusion condensates represent a newly identified category of biomolecular assemblies that play a key role in driving the oncogenic functions of RTK fusion proteins. In this study, we isolated the NACC2-NTRK2 (Nucleus Accumbens-Associated Protein 2 - Neurotrophic Receptor Tyrosine Kinase 2) fusion condensate and applied LC-MS/MS along with IMAC (Immobilized Metal Affinity Chromatography) to profile its total proteome and phospho-proteome. Our analyses revealed that the condensate selectively recruits 190 proteins via kinase activation, impacting essential cellular pathways such as signal transduction, transcriptional regulation, cell division, cell-cycle progression, and metabolic control. Fluorescence microscopy further demonstrated that proteins with distinct cellular roles interact with the condensates in different ways. Phospho-proteomic profiling showed that the kinase active condensates bring phosphorylation to 1,399 proteins and facilitate RTK crosstalk, particularly with ephrin receptor tyrosine kinases, enabling ligand-independent activation of EPHA2 (Ephrin Receptor A2) and EPHB4 (Ephrin Receptor B4).