title: Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation.
abstract: Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.
authors.affiliation: Laboratory of Molecular Regulation, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America. noriko.murakami@opwdd.ny. gov
authors.forename: Noriko
authors.initials: N
authors.lastname: Murakami
authors.forename: David C
authors.initials: DC
authors.lastname: Bolton
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kida
authors.forename: Wen
authors.initials: W
authors.lastname: Xie
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Hwang
mesh_terms: D000818:Animals; D016466:CHO Cells; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D006224:Cricetinae; D034284:Dynamin I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005260:Female; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D034041:Monomeric Clathrin Assembly Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D000097603:Dyrk Kinases
chemical_list: D034284:Dynamin I; D008869:Microtubule-Associated Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097603:Dyrk Kinases; C490546:Map1a protein, rat; C079240:Mtap2 protein, mouse; C439023:clathrin assembly protein AP180; C075637:amphiphysin; C097702:synaptojanin