Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells

Abstract

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.

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…A set of core components (e.g., dynamin and endophilin2) showed more complex scaling relationships with CCS size, perhaps reflecting variable degrees of recruitment to the budding and non-budding portions of larger CCSs. However, the recruitment signatures of a core set of proteins including GAK (a kinase essential for the uncoating reaction [16,70]), and most notably actin and actin-binding proteins, were independent of CCS size. This is consistent with our central thesis that CCVs of relatively constant size budded at host CCSs of diverse size and lifetime via a common core mechanism, and supports a role for actin in CME in NIH-3T3 fibroblasts [10,11].…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…Following scission, CCVs rapidly shed their clathrin coat through the coordinated action of the ATPase/chaperone Hsc70 (Chappell et al 1986) and neuronal J-domain kinase (Ahle and Ungewickell 1990) or ubiquitous paralog auxilin2 (cyclin-G-associated kinase, GAK) (Greener et al 2000;Umeda et al 2000).…”

Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

“…Sheep polyclonal antibody to TGN46 was purchased from Serotec (Oxford, United Kingdom), and rabbit polyclonal antibody to cathepsin D was from Calbiochem (EMD Biosciences, San Diego, CA). A rabbit polyclonal antibody to GAK was described previously (Greener et al, 2000). Donkey Alexa488-or Alexa594-conjugated anti-rabbit or anti-mouse IgG were purchased from Molecular Probes (Eugene, OR).…”

“…Here we report that the AP1-binding, cyclin G-associated kinase (GAK; also known as auxilin 2; Kanaoka et al, 1997;Kimura et al, 1997;Greener et al, 2000;Umeda et al, 2000;Lee et al, 2005Lee et al, , 2006 specifically interacts with the ear domains of AP1-␥1 and -␥2 through two sequences fitting the ⌿G [PDE][⌿LM] consensus motif. More importantly, we demonstrate that this interaction is required for the association of GAK with the TGN and for the transport of the precursor of the acid hydrolase, cathepsin D, to lysosomes.…”