Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation

• Published in: PloS one 2012-04, Vol.7 (4), p.e34845
• Main Authors: Murakami, Noriko, Bolton, David C, Kida, Elizabeth, Xie, Wen, Hwang, Yu-Wen
• Format: Article
• Language: English
• Subjects: Adaptin; Adaptor proteins; Analysis; Animals; Biology; Brain; Brain research; CHO Cells; Clathrin; Clathrin-Coated Vesicles - metabolism; Coating effects; Cricetinae; Developmental disabilities; Dissociation; Down syndrome; Dynamin; Dynamin I - metabolism; Dyrk Kinases; Electrophoresis, Polyacrylamide Gel; Endocytosis; Female; Gene expression; Hostages; Immunoglobulins; Immunology; Kinases; Laboratories; Mass spectrometry; Mice; Microscopy; Microtubule-Associated Proteins - metabolism; Monomeric Clathrin Assembly Proteins - metabolism; Mutation; Nerve Tissue Proteins - metabolism; Nervous system; Neurons; Neurosciences; PC12 Cells; Phosphatase; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Physics; Protein Serine-Threonine Kinases - metabolism; Protein-Tyrosine Kinases - metabolism; Proteins; Rats; Spectrometry; Substrates; Uncoating
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0034845

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.