Molecular basis and cellular effects of Janus-class-driven cytoplasmic PYK2 coacervates.

Kinase activity is increasingly linked to biomolecular phase separation. Focal adhesion kinase (FAK) forms membrane-associated condensates with paxillin to promote adhesion. Here we show that its paralogue, proline-rich tyrosine kinase 2 (PYK2), undergoes phase separation via a distinct mechanism. PYK2 forms cytoplasmic condensates primarily driven by its kinase-FAT linker (KFL) region. Overexpression of PYK2 induces condensates enriched in its autophosphorylated form, which sequester paxillin from focal adhesions and impair cell adhesion. We uncover an autoregulatory mechanism involving the KFL, linking self-association, autophosphorylation, and condensation. Uncommon among known phase separation drivers, KFL condensation is phosphorylation-independent and its sequence belongs to the "Janus" class. Using a transformer-based protein language model, we identified non-homologous sequences with similar features, many from adhesion and cytoskeletal regulators. We validated the phase-separating potential of several of these sequences in cells. These findings reveal a mechanism linking phase separation with kinase activation, and demonstrate distinct condensation behavior in homologs. Our results also highlight how protein concentration modulates condensate function, with implications for disease, and expand the landscape of phase separation drivers.