Phosphorylation of the rat vesicular acetylcholine transporter.
Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.
Cho
G W
GW
Department of Biochemistry, University of Kentucky, Lexington, Kentucky, 40536-0084, USA.
Kim
M H
MH
Chai
Y G
YG
Gilmor
M L
ML
Levey
A I
AI
Hersh
L B
LB
D000109:Acetylcholine / Q000378:metabolism / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000378:metabolism; D015151:Immunoblotting; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009467:Neuromuscular Depolarizing Agents / Q000493:pharmacokinetics; D016716:PC12 Cells; D010766:Phosphorylation; D010880:Piperidines / Q000493:pharmacokinetics; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D016708:Synaptophysin / Q000096:biosynthesis; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; D011994:Recombinant Proteins; C493461:Slc18a3 protein, rat; D016708:Synaptophysin; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D012694:Serine; D011493:Protein Kinase C; D000109:Acetylcholine