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

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