Liquid-Liquid Phase Separation of DDR1 Counteracts the Hippo Pathway to Orchestrate Arterial Stiffening

Abstract

Background: The Hippo-YAP (yes-associated protein) signaling pathway is modulated in response to various environmental cues. Activation of YAP in vascular smooth muscle cells conveys the extracellular matrix stiffness-induced changes in vascular smooth muscle cells phenotype and behavior. Recent studies have established a mechanoreceptive role of receptor tyrosine kinase DDR1 (discoidin domain receptor 1) in vascular smooth muscle cells. Methods: We conduced 5/6 nephrectomy in vascular smooth muscle cells-specific Ddr1-knockout mice, accompanied by pharmacological inhibition of the Hippo pathway kinase LATS1 (large tumor suppressor 1), to investigate DDR1 in YAP activation. We utilized polyacrylamide gels of varying stiffness or the DDR1 ligand, type I collagen, to stimulate the cells. We employed multiple molecular biological techniques to explore the role of DDR1 in controlling the Hippo pathway and to determine the mechanistic basis by which DDR1 exerts this effect. Results: We identified the requirement for DDR1 in stiffness/collagen-induced YAP activation. We uncovered that DDR1 underwent stiffness/collagen binding-stimulated liquid-liquid phase separation and co-condensed with LATS1 to inactivate LATS1. Mutagenesis experiments revealed that the transmembrane domain is responsible for DDR1 droplet formation. Purified DDR1 N-terminal and transmembrane domain was sufficient to drive its reversible condensation. Depletion of the DDR1 C-terminus led to failure in co-condensation with LATS1. Interaction between the DDR1 C-terminus and LATS1 competitively inhibited binding of MOB1 (Mps one binder 1) to LATS1 and thus the subsequent phosphorylation of LATS1. Introduction of the single-point mutants, histidine-745-proline and histidine-902-proline, to DDR1 on the C-terminus abolished the co-condensation. In mouse models, YAP activity was positively correlated with collagen I expression and arterial stiffness. LATS1 inhibition reactivated the YAP signaling in Ddr1-deficient vessels and abrogated the arterial softening effect of Ddr1 deficiency. Conclusions: These findings identify DDR1 as a mediator of YAP activation by mechanical and chemical stimuli and demonstrate that DDR1 regulates LATS1 phosphorylation in an liquid-liquid phase separation-dependent manner.