Dynamic Phosphoproteomics of BRS3 Activation Reveals the Hippo Signaling Pathway for Cell Migration

Bombesin receptor subtype-3 (BRS3) is an orphan G-proteincoupledreceptor (GPCR) that is involved in a variety of pathological andphysiological processes, while its biological functions and underlyingregulatory mechanisms remain largely unknown. In this study, a quantitativephosphoproteomics approach was employed to comprehensively decipherthe signal transductions that occurred upon intracellular BRS3 activation.The lung cancer cell line H1299-BRS3 was treated with MK-5046, anagonist of BRS3, for different durations. Harvested cellular proteinswere digested and phosphopeptides were enriched by immobilized titanium(IV) ion affinity chromatography (Ti4+-IMAC) for label-freequantification (LFQ) analysis. A total of 11,938 phosphopeptides wereidentified, corresponding to 3,430 phosphoproteins and 10,820 phosphosites.Data analysis revealed that 27 phosphopeptides corresponding to sixproteins were involved in the Hippo signaling pathway, which was significantlyregulated by BRS3 activation. Verification experiments demonstratedthat downregulation of the Hippo signaling pathway caused by BRS3activation could induce the dephosphorylation and nucleus localizationof the Yes-associated protein (YAP), and its association with cellmigration was further confirmed by kinase inhibition. Our data collectivelydemonstrate that BRS3 activation contributes to cell migration throughdownregulation of the Hippo signaling pathway.

Automated summary

In this study, a quantitative phosphoproteomics approach was used to investigate the signal transductions upon intracellular BRS3 activation. The results revealed that BRS3 activation downregulates the Hippo signaling pathway, leading to cell migration.