Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events

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.