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.
Highlights
Internalization from the plasma membrane occurs by different pathways, including uptake via clathrin-coated vesicles (CCVs)
The major endocytic CCV constituents, clathrin and AP-2, are functionally regulated by phosphorylation, as well as other factors including dynamin 1, amphiphysins, synaptojanin, AP180, epsin, and eps15, which are collectively grouped as dephosphins, that become active in their dephosphorylated state [2]
Tyrosine residues have been identified as phosphorylation target sites and serine/threonine kinases such as the recently identified CVAK104 [14] may impact on the phosphorylation status of 2-adaptin
Summary
Internalization from the plasma membrane occurs by different pathways, including uptake via clathrin-coated vesicles (CCVs).4 it is obvious that clathrin coat assembly and disassembly need to be tightly regulated both spatially and tem-. 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.
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