A novel AAK1 splice variant functions at multiple steps of the endocytic pathway.
Phosphorylation is a critical step in regulating receptor transport through the endocytic pathway. AAK1 is a serine/threonine kinase that is thought to coordinate the recruitment of AP-2 to receptors containing tyrosine-based internalization motifs by phosphorylating the micro2 subunit. Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin. However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 micro2 subunit. However, overexpression of AAK1L CBD2 impairs transferrin endocytosis, confirming an endocytic role for AAK1. Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome. These observations suggest that AAK1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its rapid recycling back to the plasma membrane from early/sorting endosome.
Henderson
Davin M
DM
Department of Genetics, University of Minnesota, Minneapolis, MN 55455, USA.
Conner
Sean D
SD
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001921:Brain / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D002966:Clathrin; D007527:Isoenzymes; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D014168:Transferrin; C451304:AAK1 protein, human; D017346:Protein Serine-Threonine Kinases