Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation, and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Since macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent cell survival through the incorporation of extracellular nutrients.

This study reveals a novel role of d-amino acid oxidase in promoting cellular senescence induced by genotoxic stresses via enzymatic generation of reactive oxygen species.

Cellular senescence is a highly stable cell-cycle arrest induced by DNA damage and various cellular stresses. Recently, we have revealed that lymphocyte antigen 6 complex, locus D (LY6D) is responsible for senescence-inducing stress-evoked vacuole formation through induction of Src family kinase (SFK)-mediated macropinocytosis. However, the signaling molecule(s) transducing the macropinocytosis signal from extracellular LY6D to the cytoplasmic SFK are unknown. In this study, we identified integrin b1, a transmembrane signaling protein, as an interactor of LY6D by proteomic analysis and co-immunoprecipitation assays. Inhibition of integrin b1 impaired LY6D-induced macropinocytosis, and integrin b1 activated SFK through focal adhesion kinase to mediate macropinocytosis. These results indicate that integrin b1 is a crucial mediator of the LY6D-induced vacuole formation in senescent cells.