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.