Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation.

Abstract

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the $#x03BC;2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of $#x03BC;2 phosphorylation demonstrated that clathrin is a specific activator of the $#x03BC;2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of $#x03BC;2 phosphorylation. Furthermore, phosphorylated $#x03BC;2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-$#x03BC;2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.

Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry

“…Cells transfected with AP-2␣-wt-EGFP were allowed to express the construct for 24 to 48 h before the start of the experiment. The prolonged expression time is required for efficient incorporation of the GFP-labeled ␣ subunit into the endogenous adaptor protein complex (16 (20,80, or 120 M) (ASINEX), brefeldin A (1 M) (Sigma-Aldrich), nocodazole (1 M) (Sigma-Aldrich), or wortmannin (0.1, 1, or 10 M) (Sigma-Aldrich). Due to the high cell toxicity of chlorpromazine (SigmaAldrich), cells were preincubated with 17, 70, 140, or 280 M of the drug for 10 min before the virus was added, and the drug-containing inoculum was left on the cells for 30 min before replacement with complete medium.…”

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

“…The dephosphins are found in the phosphorylated state in resting nerve terminals and are coordinately dephosphorylated upon stimulation of the nerve terminals (Cousin and Robinson, 2001). In addition, the clathrin heavy chain and one of the clathrin LCs (LCb) as well as the large and the medium subunits of AP1 and AP2 are subject to phosphorylation both in vitro and in vivo (Bar-Zvi and Branton, 1986;Flett et al, 2005). It is known that some of the kinases responsible for the phosphorylation mentioned above are associated with purified CCVs.…”

Characterization of a Protein Phosphatase 2A Holoenzyme That Dephosphorylates the Clathrin Adaptors AP-1 and AP-2

“…Because AP-2 is a key factor in the formation of CCVs and known for a long time to be phosphorylated, it is an ideal model protein to analyze the functional consequences and regulatory mechanisms of cycles of phosphorylation (3). The complex is phosphorylated on the medium subunit 2 at a single threonine residue (Thr-156), which is mediated in vitro by the two kinases AAK1 and GAK (4 -8).…”