In vivo phosphorylation of adaptors regulates their interaction with clathrin.
The coat proteins of clathrin-coated vesicles (CCV) spontaneously self-assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly.
G.W. Hooper Foundation, Department of Immunology and Microbiology, University of California, San Francisco 94143-0552, USA.
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Wilde
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Brodsky
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002966:Clathrin / Q000096:biosynthesis / Q000378:metabolism*; D018699:Coated Vesicles / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010770:Phosphotransferases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008565:Membrane Proteins; D010446:Peptide Fragments; D010768:Phosphoserine; D010770:Phosphotransferases; D000469:Alkaline Phosphatase