Dyrk1A binds to multiple endocytic proteins required for formation of clathrin-coated vesicles.
In spite of a nuclear targeting sequence, a substantial amount of dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk1A) is located within the cytoplasm of neurons. Analysis of fractionated rat brains revealed that the majority of Dyrk1A was in the postnuclear precipitate. The kinase in this fraction was resistant to high salt and Triton X-100 extraction at pH 6.5. Hypothesizing that Dyrk1A binds tightly with cell constituents, we searched for Dyrk1A binding proteins in the Triton X-100-insoluble fraction extracted with urea and fractionated by column chromatography. An overlay assay using the recombinant kinase revealed that multiple proteins are capable of binding to Dyrk1A. Among them, we identified clathrin heavy chain and dynamin 1 as potential candidates. An overlay assay using purified and partially purified proteins showed the binding of Dyrk1A with both proteins. Under native conditions, Dyrk1A precipitated with newly formed clathrin cages and with dynamin via the GST-amphiphysin SH3 domain. We also identified another endocytic protein, endophilin 1, as an additional Dyrk1A binding protein. We then tested whether the clathrin-coated vesicle (CCV)-associated proteins could be phosphorylated by Dyrk1A. Multiple proteins apparently distinctive from the known substrates were phosphorylated in the brain CCV. Our findings suggest a role for Dyrk1A in controlling synaptic vesicle recycling processes.
Murakami
Noriko
N
Laboratory of Molecular Regulations, Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. Noriko.Murakami@omr.state
Bolton
David
D
Hwang
Yu-Wen
YW
D000818:Animals; D001692:Biological Transport; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000201:enzymology / Q000378:metabolism*; D003956:Dialysis; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D022161:Transport Vesicles / Q000201:enzymology / Q000378:metabolism*; D000097603:Dyrk Kinases
D002352:Carrier Proteins; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097603:Dyrk Kinases