The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Abstract

Phosphatidylinositol 3-phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain-containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid-phase marker dextran were retained in abnormally small endosomes in Phafin2-depleted cells. In yeast two-hybrid analysis we identified Phafin2 as a novel interactor of the endosomal-tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid-phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.

SNX15 links clathrin endocytosis to the PtdIns(3)P early endosome independent of the APPL1 endosome

“…More recently, genomewide (Collinet et al, 2010) and targeted (Pedersen et al, 2012) RNAi screens have independently identified SNX15 as a regulator of degradative EGFR sorting, although the details of its role have yet to be described [the latter screen specifically targeted the human proteome for proteins that contain phosphoinositide-binding PX and FYVE domains (Pedersen et al, 2012)]. Human SNX15 is a 342 amino acid protein composed of a central region that includes a proline-rich stretch of three PPPxxP repeats (residues P142 to P181) and an acidic cluster (E 255 EEEDGE 261 ), flanked by an N-terminal SNX-PX domain (F9 to G126) and a C-terminal microtubule-interacting and trafficking (MIT) domain (A267 to P342; Fig.…”

WDFY2 restrains matrix metalloprotease secretion and cell invasion by retention of VAMP3 in endosomal tubules

“…mRNA expression analysis was done as described in Pedersen et al, 2012 48 . The primers used in the experiment were QT00035455 for WDFY2 and QT00000721 for TBP for housekeeping (Qiagen).…”

The class III phosphatidylinositol 3-kinase Vps34 in Saccharomyces cerevisiae

“…The subsequent signaling cascades are involved in endocytic transport and endocytic signaling, cytokinesis as well as autophagy (Simonsen and Tooze, 2009;Funderburk et al, 2010;Nezis et al, 2010;Platta and Stenmark, 2011;Pedersen et al, 2012;Schink et al, 2016). In humans, these PIK3C3-dependent processes have a central function in the prevention of tumorigenesis, by ensuring the termination of growth factor receptor signaling, as well as by the engulfment of damaged organelles in autophagosomes, or through prevention of bi-nucleation and genome instability by promoting a correct cytokinesis (Stenmark, 2010;Morris et al, 2015).…”