Analysis of FYVE Domain-Containing Proteins in Signaling and Endocytosis: a dissertation

Abstract

The FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.