The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-β/Smad signalling

Background: Transforming growth factor-beta (TGF-beta) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF-beta type I (TbetaR-I) and TGF-beta type II serine/threonine kinase receptors (TbetaR-II). After the activation of TbetaR-I kinase by TbetaR-II kinase, specific receptor-regulated Smads (R-Smads) are phosphorylated by TbetaR-I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R-Smads and presents them to TbetaR-I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain. Results: Wild-type SARA and the FYVE domain of SARA (FYVE(SARA)) reveal a punctate staining pattern and co-localize with the early endosomal markers, early endosomal antigen-1 (EEA1) and hepatic growth factor-regulated tyrosine kinase substrate (Hrs). The ectopic expression of dominant negative rab5, a critical regulatory molecule in endosome function, redistributes SARA from punctate to a diffuse cytosolic staining pattern. A lipid binding assay demonstrated that the recombinant FYVE domain from SARA predominantly interacts with phosphatidylinositol 3-phosphate (PtdIns(3)P). Consistent with this result, wortmannin, a PI3 kinase inhibitor, resulted in both a redistribution of SARA from the endosomal compartment to the cytosol and the attenuation of both TGF-beta-induced R-Smad phosphorylation and transcriptional activation. Ectopic expression of the FYVE domain of SARA also induced the redistribution of wild-type SARA and inhibited TGF-beta as well as BMP/Smad-induced transcriptional responses. Conclusion: The FYVE domain is sufficient and necessary for the early endosomal localization of SARA, probably through its interaction with PtdIns(3)P Moreover, the localization of SARA in early endosomes is required for efficient TGF-beta/ Smad signalling.