Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

Abstract

1 Spatio-temporal regulation of central adaptor complex, AP-2 is pivotal for clathrin-mediated endocytosis 2 (CME). We recently discovered that FCHO proteins trigger clathrin-coated pit (CCP) formation by 3 allosterically activating AP-2 on plasma membrane (Umasankar et al., 2014). Here, we demonstrate that 4 this activation promotes AP-2 phosphorylation via recruitment and stabilization of BMP-2 inducible 5 kinase (BMP2K), a bona fide AP-2 kinase leading to CCP maturation. Accordingly, BMP2K mislocalizes 6 and degrades in FCHO knockout/ AP-2 depleted cells. Functional inactivation of kinase impairs AP-2 7 phosphorylation leading to altered lattice morphology and CME phenotypes reminiscent of CCP 8 maturation defects. Reexpression of FCHO rescues AP-2 phosphorylation defects in FCHO knockout 9 cells implying membrane activation of AP-2 is a prerequisite for kinase function. Furthermore, gain-and 10 loss-of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions 11 during zebrafish embryogenesis. Together, our findings reveal an in vivo allosteric feed-forward axis for 12 operation of CME. 13