Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis.
The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.
Conner
Sean D
SD
The Scripps Research Institute, La Jolla, CA 92037, USA.
Schmid
Sandra L
SL
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence / Q000502:physiology; D064127:Aurora Kinases; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003001:Cloning, Molecular; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004705:Endocytosis / Q000502:physiology*; D006367:HeLa Cells; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D018076:DNA, Complementary; D008565:Membrane Proteins; C061570:protein kinase N; C451304:AAK1 protein, human; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C