T. Greener et al., Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells, J BIOL CHEM, 275(2), 2000, pp. 1365-1370
Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to diss
ociate 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 r
equired 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, W
estern blot analysis shows that GAK is present in rat liver, bovine testes,
and bovine brain clathrin-coated vesicles. More importantly, liver clathri
n-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 clat
hrin polymerization into baskets and the combined clathrin-binding domain a
nd 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, Immunolocaliza
tion studies suggest that GAK is a cytosolic protein that is concentrated i
n the perinuclear region; it appears to be highly associated with the trans
-Golgi where the budding of clathrin-coated vesicles occurs. We propose tha
t GAK is a required cofactor for the uncoating of clathrin-coated vesicles
by Hsc70 in non-neuronal cells.