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 (α- and β2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit μ2. Phosphorylation of μ2 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 μ2, ∼25 min for β2, and ∼70 min for α. We were also able to purify a phosphatase that dephosphorylates the μ2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cβ subunit, a scaffolding Aβ subunit, and a regulatory Bα 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.