title: Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2.
abstract: The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.
authors.affiliation: Department of Biomedical Science and Biotechnology, University of Brescia, 25100 Brescia, Italy.
authors.forename: Doris
authors.initials: D
authors.lastname: Ricotta
authors.forename: Jens
authors.initials: J
authors.lastname: Hansen
authors.forename: Carolin
authors.initials: C
authors.lastname: Preiss
authors.forename: Dominic
authors.initials: D
authors.lastname: Teichert
authors.forename: Stefan
authors.initials: S
authors.lastname: Höning
mesh_terms: D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D033966:Adaptor Protein Complex beta Subunits / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006367:HeLa Cells; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013552:Swine
chemical_list: D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D002966:Clathrin; D020035:Holoenzymes; D054648:Protein Phosphatase 2