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 beta 2- adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu 2. Phosphorylation of mu 2 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 mu 2, similar to 25 min for beta 2, and similar to 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu 2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic C beta subunit, a scaffolding A beta subunit, and a regulatory B alpha 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.