Phosphorylation of a vesicular monoamine transporter by casein kinase II.
The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.
Krantz
D E
DE
Department of Neurology, University of California School of Medicine, San Francisco, California 94143-0435, USA.
Peter
D
D
Liu
Y
Y
Edwards
R H
RH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D019556:COS Cells; D047390:Casein Kinase II; D047388:Casein Kinases; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009479:Neuropeptides; D016716:PC12 Cells; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013329:Structure-Activity Relationship; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C493452:SLC18A2 protein, human; C493451:Slc18a1 protein, rat; C493454:Slc18a2 protein, rat; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D010768:Phosphoserine; D012701:Serotonin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases