Multiple Roles for Cyclin G‐Associated Kinase in Clathrin‐Mediated Sorting Events

Abstract

Cyclin G-associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin-mediated membrane trafficking. It possesses a kinase domain at its N-terminus that can phosphorylate the clathrin adaptors AP-1 and AP-2 in vitro. The GAK C-terminus can act as a cochaperaone in vitro for Hsc70, a heat-shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor-associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAK's in vivo function. We discovered that early stages of clathrin-mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin-dependent trafficking from the trans Golgi network.

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

“…In agreement with previous reports (Greener et al, 2000;Zhang et al, 2005;Lee et al, 2006), immunofluorescence microscopy analyses showed that a fraction of endogenous GAK colocalized with TGN46 ( Figure 6, A-C), clathrin ( Figure 6, D-F), and AP1 (Figure 6, G-I) at the TGN. Treatment with brefeldin A (BFA), which dissociates Arf1-regulated coat proteins such as AP1 from membranes (Robinson and Kreis, 1992;Wong and Brodsky, 1992), redistributed GAK to the cytosol ( Figure 6, J-L).…”

“…Although it is now clear that both the expression of GAK (Lee et al, 2005;Zhang et al, 2005; this study) and its ability to interact with AP1 (this study) are required for lysosomal enzyme sorting, the exact mechanism by which GAK participates in this process remains to be elucidated. GAK contains a Ser/Thr kinase domain homologous to that of AAK1, an enzyme that phosphorylates the 2 subunit of AP2 and activates it for recognition of YXXØ-type sorting signals Ricotta et al, 2002;Honing et al, 2005).…”

“…Previous studies showed that GAK is enriched in plasma membrane and TGN clathrin coats (Greener et al, 2000;Lee et al, 2006), where it phosphorylates the AP2-2 and AP1-1 subunits, respectively (Umeda et al, 2000;Korolchuk and Banting, 2002). In addition, GAK was shown to promote clathrin uncoating, thus regulating receptor endocytosis at the plasma membrane (Greener et al, 2000;Zhao et al, 2001;Zhang et al, 2004;Lee et al, 2005) and sorting of MPRs and cathepsin D at the TGN (Lee et al, 2005;Zhang et al, 2005). These properties of GAK are consistent with the presence of putative ␥-ear-and GGA-GAE-binding motifs within its sequence.…”

“…The interaction of GAK with AP1 described here thus likely enables the recruitment of both the kinase and uncoating activity of GAK to sites of lysosomal enzyme sorting. In vitro kinase assay using GFP alone and GFP fused to wild-type (wt), AP1-␥1-ear-binding site mutant (mut1/2) or kinase-deficient (K69A; Zhang et al, 2005) GAK. GFP proteins were expressed by transfection in HeLa cells, immunoprecipitated with anti-GFP antibody, and analyzed using an in vitro kinase assay with myelin basic protein (MBP) as a substrate, as described in Materials and Methods.…”

Scd5p Mediates Phosphoregulation of Actin and Endocytosis by the Type 1 Phosphatase Glc7p in Yeast

“…Interestingly, AAK1 shares with Prk1p not only the extensive kinase sequence homology but also the similar recognition motif Ricotta et al, 2002). Another mammalian homologue of Prk1p, GAK, also exhibits a similar specificity on the 2 subunit and plays a regulatory role in endocytosis (Zhang et al, 2005). AP2 is known to associate with Eps15, the mammalian counterpart of Pan1p, and such interaction is required for endocytosis (Benmerah et al, 1995(Benmerah et al, , 1998.…”