Phosphorylation of Amphiphysin I by Minibrain Kinase/Dual-specificity Tyrosine Phosphorylation-regulated Kinase, a Kinase Implicated in Down Syndrome

Noriko Murakami,Wen Xie,Renne Chen Lu,Mo-Chou Chen-Hwang,Andrzej Wieraszko,Yu Wen Hwang

doi:10.1074/jbc.m513497200

Abstract

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.

Highlights

After exocytosis of neurotransmitters, rapid endocytosis for recycling synaptic vesicle constituents occurs via clathrin-mediated endocytosis
Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of amphiphysin I (Amph I) at Serine 293 (Ser-293) and that phosphorylation changes the recruitment of endophilin at the endocytic sites
We speculated that human Amph I could be phosphorylated by Mnb/Dyrk1A

Summary

EXPERIMENTAL PROCEDURES

Materials—Nerve growth factor (NGF), LipofectAmine (PLUS and 2000), fetal bovine serum (FBS), horse serum, Dulbecco’s modified Eagle’s medium, and antibiotic mixtures were from Invitrogen. Expression and purification of recombinant Mnb/ Dyrk1A and glutathione S-transferase (GST)-endophilin 1 were described previously [19]. Truncated Amph I was expressed either as GST fusion by ligating each DNA fragment between the ClaI and XhoI sites of pGEX or as poly-His-tagged proteins at their C terminus (12 histidine repeat) by reconstruction in the pND vector [23]. GST-PRD or PRD-poly-His was purified from E. coli by extracting the cell pellets with a buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM imidazole (pH 7.4), 5 mM 2-mercaptoethanol, 1%Triton X-100, and 0.15 M NaCl followed by purification using either glutathione (GT) or nickel nitrilotriacetic acid resins. PC-12 cells were grown over rat collagen-coated culture dishes in F-12K medium containing 12.5% horse serum and 2.5% FBS

RESULTS

Ser Ͼ Thr Ser ND ND

DISCUSSION