title: 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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Claire X
authors.initials: CX
authors.lastname: Zhang
authors.forename: Asa E Y
authors.initials: AE
authors.lastname: Engqvist-Goldstein
authors.forename: Sebastien
authors.initials: S
authors.lastname: Carreno
authors.forename: David J
authors.initials: DJ
authors.lastname: Owen
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Smythe
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
mesh_terms: D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002966:Clathrin / Q000502:physiology*; D016213:Cyclins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051381:Rats; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D002966:Clathrin; D016213:Cyclins; C103904:Gak protein, rat; D014168:Transferrin; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D002402:Cathepsin D