Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome.
Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.
Murakami
Noriko
N
Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. Noriko.Murakami@omr.state.ny.us
Xie
Wen
W
Lu
Renne Chen
RC
Chen-Hwang
Mo-Chou
MC
Wieraszko
Andrzej
A
Hwang
Yu Wen
YW
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001666:Binding Sites, Antibody; D016466:CHO Cells; D006224:Cricetinae; D004314:Down Syndrome / Q000201:enzymology* / Q000378:metabolism; D004594:Electrophysiology; D004705:Endocytosis / Q000235:genetics; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011505:Protein-Tyrosine Kinases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D000097603:Dyrk Kinases
D048868:Adaptor Proteins, Signal Transducing; D009419:Nerve Tissue Proteins; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012694:Serine; D000097603:Dyrk Kinases; C107640:SH3GL2 protein, human; C075637:amphiphysin