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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Substitution, Animals, Binding Sites, Antibody, CHO Cells, Cricetinae, Down Syndrome, Electrophysiology, Endocytosis, Hippocampus, Humans, Mice, NIH 3T3 Cells, Nerve Tissue Proteins, Organ Culture Techniques, Phosphorylation, Proline, Protein Binding, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Rats, Recombinant Fusion Proteins, Serine
Adaptor Proteins, Signal Transducing, Amino Acid Substitution, Animals, Binding Sites, Antibody, CHO Cells, Cricetinae, Down Syndrome, Electrophysiology, Endocytosis, Hippocampus, Humans, Mice, NIH 3T3 Cells, Nerve Tissue Proteins, Organ Culture Techniques, Phosphorylation, Proline, Protein Binding, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Rats, Recombinant Fusion Proteins, Serine
J. Biol. Chem.
Date: Aug. 18, 2006
PubMed ID: 16733250
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