Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells.
Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.
Greener
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Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0301, USA.
Zhao
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Nojima
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Eisenberg
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Greene
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D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002966:Clathrin / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013737:Testis / Q000201:enzymology; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D016213:Cyclins; C103904:Gak protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; C486325:GAK protein, human; D017346:Protein Serine-Threonine Kinases