Dataset Information

A novel AAK1 splice variant functions at multiple steps of the endocytic pathway.

ABSTRACT: 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.

SUBMITTER: Henderson DM

PROVIDER: S-EPMC1924820 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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A novel AAK1 splice variant functions at multiple steps of the endocytic pathway.

Henderson Davin M DM Conner Sean D SD

Molecular biology of the cell 20070509 7

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 i ...[more]

PMID: 17494869

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Project description:Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Dataset Information

A novel AAK1 splice variant functions at multiple steps of the endocytic pathway.

Publications

A novel AAK1 splice variant functions at multiple steps of the endocytic pathway.

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