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.

[1]  P. De Camilli,et al.  The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals* , 1999, The Journal of Biological Chemistry.

[2]  L. Hersh,et al.  Mutational Analysis of Aspartate Residues in the Transmembrane Regions and Cytoplasmic Loops of Rat Vesicular Acetylcholine Transporter* , 1999, The Journal of Biological Chemistry.

[3]  L. Hersh,et al.  The Cholinergic Gene Locus Is Coordinately Regulated by Protein Kinase A II in PC12 Cells , 1998, Journal of neurochemistry.

[4]  P. De Camilli,et al.  Role of phosphorylation in regulation of the assembly of endocytic coat complexes. , 1998, Science.

[5]  R. Edwards,et al.  A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters* , 1998, The Journal of Biological Chemistry.

[6]  J. Erickson,et al.  The Cytoplasmic Tail of the Vesicular Acetylcholine Transporter Contains a Synaptic Vesicle Targeting Signal* , 1998, The Journal of Biological Chemistry.

[7]  V. Prado,et al.  Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol , 1997, Journal of neurochemistry.

[8]  S. Hunt,et al.  Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis. , 1997, Molecular biology of the cell.

[9]  Jun-Ping Liu,et al.  PROTEIN PHOSPHORYLATION EVENTS IN EXOCYTOSIS AND ENDOCYTOSIS , 1997, Clinical and experimental pharmacology & physiology.

[10]  C. Geisler,et al.  Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro , 1997, The Journal of cell biology.

[11]  M. Poo,et al.  Expression of a Putative Vesicular Acetylcholine Transporter Facilitates Quantal Transmitter Packaging , 1997, Neuron.

[12]  R. Edwards,et al.  Phosphorylation of a Vesicular Monoamine Transporter by Casein Kinase II* , 1997, The Journal of Biological Chemistry.

[13]  J. Erickson,et al.  Active Transport of Acetylcholine by the Human Vesicular Acetylcholine Transporter* , 1996, The Journal of Biological Chemistry.

[14]  Amy Johnson,et al.  The Carboxyl Terminus of GLUT4 Contains a Serine-Leucine-Leucine Sequence That Functions as a Potent Internalization Motif in Chinese Hamster Ovary Cells* , 1996, The Journal of Biological Chemistry.

[15]  H. Yi,et al.  Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[16]  P. Camilli,et al.  A presynaptic inositol-5-phosphatase , 1996, Nature.

[17]  M. Jackson,et al.  A Role for Acidic Residues in Di-leucine Motif-based Targeting to the Endocytic Pathway (*) , 1995, The Journal of Biological Chemistry.

[18]  T. Bonner,et al.  Molecular biology of the vesicular ACh transporter , 1995, Trends in Neurosciences.

[19]  L. Hersh,et al.  Expression of the Choline Acetyltransferase Gene Depends on Protein Kinase A Activity , 1995, Journal of neurochemistry.

[20]  S. Parsons,et al.  Effects of Internal pH on the Acetylcholine Transporter of Synaptic Vesicles , 1995, Journal of neurochemistry.

[21]  R. Edwards,et al.  Molecular cloning of a putative vesicular transporter for acetylcholine. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[22]  T. Bonner,et al.  Functional identification of a vesicular acetylcholine transporter and its expression from a "cholinergic" gene locus. , 1994, The Journal of biological chemistry.

[23]  T. Bonner,et al.  Cloning and expression of the vesamicol binding protein from the marine ray Torpedo , 1994, FEBS letters.

[24]  Li Yang,et al.  Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) , 1994, Molecular reproduction and development.

[25]  L. Pinna,et al.  Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinases-1 and -2. , 1994, Biochemical and biophysical research communications.

[26]  B. Hoflack,et al.  Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor. , 1993, The Journal of biological chemistry.

[27]  A. Alfonso,et al.  The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter. , 1993, Science.

[28]  B. Hoffman,et al.  Expression cloning of a reserpine-sensitive vesicular monoamine transporter. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[29]  S. Kornfeld,et al.  The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi , 1992, The Journal of cell biology.

[30]  D. Eisenberg,et al.  A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter , 1992, Cell.

[31]  R. Klausner,et al.  A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains , 1992, Cell.

[32]  P. De Camilli,et al.  Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons , 1992, The Journal of cell biology.

[33]  D. Ginty,et al.  Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities. , 1991, The Journal of biological chemistry.

[34]  J. Zheng,et al.  Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[35]  M. Crumpton,et al.  A method for measuring protein kinase C activity in permeabilized T lymphocytes by using peptide substrates. Evidence for multiple pathways of kinase activation. , 1990, The Biochemical journal.

[36]  B. Kemp,et al.  Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. , 1987, Science.

[37]  R. Weinmann,et al.  Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription. , 1986, The Journal of biological chemistry.

[38]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[39]  S. Parsons,et al.  Acetylcholine transport, storage, and release. , 1993, International review of neurobiology.

[40]  R. Pearson,et al.  Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. , 1991, Methods in enzymology.

[41]  T. Kunkel,et al.  Efficient site-directed mutagenesis using uracil-containing DNA. , 1991, Methods in enzymology.

[42]  P. De Camilli,et al.  Pathways to regulated exocytosis in neurons. , 1990, Annual review of physiology.

[43]  G. Rudnick.  ATP-driven H+ pumping into intracellular organelles. , 1986, Annual review of physiology.