title: Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase.
abstract: The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including "ecstasy" (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N(6)-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-(33)P]ATP but not from similarly labeled N(6)-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Wong
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.forename: Grace R
authors.initials: GR
authors.lastname: Jeschke
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.forename: Gary
authors.initials: G
authors.lastname: Rudnick
mesh_terms: D019943:Amino Acid Substitution; D001693:Biological Transport, Active / Q000502:physiology; D006152:Cyclic GMP / Q000235:genetics / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D012701:Serotonin / Q000235:genetics / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D048051:p38 Mitogen-Activated Protein Kinases; D006152:Cyclic GMP