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.
Zhang
Claire X
CX
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
Engqvist-Goldstein
Asa E Y
AE
Carreno
Sebastien
S
Owen
David J
DJ
Smythe
Elizabeth
E
Drubin
David G
DG
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
D002966:Clathrin; D016213:Cyclins; C103904:Gak protein, rat; D014168:Transferrin; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D002402:Cathepsin D