title: Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin.
abstract: Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.
authors.affiliation: Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: Hongying
authors.initials: H
authors.lastname: Shen
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Ferguson
authors.forename: Noah
authors.initials: N
authors.lastname: Dephoure
authors.forename: Ryan
authors.initials: R
authors.lastname: Park
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Laura
authors.initials: L
authors.lastname: Volpicelli-Daley
authors.forename: Steven
authors.initials: S
authors.lastname: Gygi
authors.forename: Joseph
authors.initials: J
authors.lastname: Schlessinger
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics* / Q000378:metabolism*; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D033941:Clathrin Light Chains / Q000235:genetics / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D034284:Dynamin I / Q000235:genetics* / Q000378:metabolism; D034285:Dynamin II / Q000235:genetics* / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D005347:Fibroblasts; D055786:Gene Knockout Techniques; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D002966:Clathrin; D033941:Clathrin Light Chains; D033922:Clathrin Heavy Chains; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; D034284:Dynamin I; D020764:cdc42 GTP-Binding Protein; D034285:Dynamin II