title: AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation.
abstract: The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
authors.affiliation: Washington University School of Medicine, Department of Internal Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110, USA.
authors.forename: Pradipta
authors.initials: P
authors.lastname: Ghosh
authors.forename: Stuart
authors.initials: S
authors.lastname: Kornfeld
mesh_terms: D033961:Adaptor Protein Complex 1 / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D020134:Catalytic Domain / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007739:L Cells; D008024:Ligands; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D054648:Protein Phosphatase 2; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*
chemical_list: D033961:Adaptor Protein Complex 1; D004791:Enzyme Inhibitors; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D008024:Ligands; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2