Reciprocal regulation of endocytosis and signaling in Drosophila epithelial development and aberrant growth

Abstract

Endocytosis and subsequent endosomal sorting of plasma membrane proteins and nutrients are essential for cellular homeostasis. Previous work has uncovered several different internalization mechanisms, endosomal sorting routes and aspects of their regulation. Endocytosis components have also been variably implicated in signaling activation or silencing, but the underlying mechanisms of how endocytosis and signaling are coordinated in epithelial tissues remain obscure. We have characterized an endocytic regulatory circuit, where type III receptor tyrosine phosphatases (RPTPs) antagonize the non-receptor tyrosine kinase Btk29A in phosphorylating the actin nucleation promoting factor WASH. Btk29A phosphorylates a conserved tyrosine motif in WASH (Y273) and this modification is sufficient to induce endosomal actin polymerization both in Drosophila airways and in mammalian cultured cells. The phospho-WASH-induced actin polymerization promotes apical protein internalization and destabilizes cortical actin altering epithelial cell and organ shapes. To investigate the function of the Ptp10D/Btk29A/WASH circuit in the context of proliferating cells, we turned to a model of tumor-suppressive cell competition in Drosophila imaginal eye discs. Clones of polarity-deficient (scrib-) epithelial cells are normally eliminated from the eye discs but overgrow when Ptp10D is also inactivated in the clones. We found that constitutive WASH phosphorylation on Y273 is sufficient to induce Yki activation and epidermal growth factor receptor (EGFR) signaling, leading to clone overgrowth. To understand how endosomal phospho-WASH may induce EGFR signaling, we investigated the endocytic routes of EGFR and Ptp10D. EGFR is recycled from endosomes to the plasma membrane by the retriever/WASH complex but Ptp10D utilizes the retromer/WASH complex. The dysregulated WASH phosphorylation results in the separation of EGFR from its inhibitor, Ptp10D, in endosomes leading to EGFR signaling upregulation and aberrant clonal growth. We showed that WASH phosphorylation is regulated in the retromer- enriched microdomain by the Stit and Insulin receptors, which antagonize Ptp10D. In turn, retromer function is required for Stit recycling suggesting a positive feedback loop between metabolism and retromer trafficking through WASH phosphorylation. Imbalances in this regulatory loop can lead to the overgrowth of polarity-deficient epithelial cells.

Keywords: endocytosis, protein sorting, RTK signaling, airway maturation, tumor-suppressive cell competition.

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