Characterization of intracellular regions in the human serotonin transporter for phosphorylation sites.
In the central nervous system, synaptic levels of the monoamine neurotransmitter serotonin are mainly controlled by the serotonin transporter (SERT), and drugs used in the treatment of various psychiatric diseases have SERT as primary target. SERT is a phosphoprotein that undergoes phosphorylation/dephosphorylation during transporter regulation by multiple pathways. In particular, activation and/or inhibition of kinases including PKC, PKG, p38MAPK, and CaMKII modulate SERT function and trafficking. The molecular mechanisms by which kinase activity is linked to SERT regulation are poorly understood, including the identity of specific phosphorylated residues. To elucidate SERT phosphorylation sites, we have generated peptides corresponding to the entire intracellular region of human SERT and performed in vitro phosphorylation assays with a panel of kinases suggested to be involved in SERT regulation or for which canonical phosphorylation sites are predicted. Peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and quantify site-specific phosphorylation. Five residues located in the N- and C-termini and in intracellular loop 1 and 2 were identified as phosphorylation sites; Ser149, Ser277, and Thr603 for PKC, Ser13 for CaMKII, and Thr616 for p38MAPK. Possible regulatory roles of these potential phosphoacceptors for SERT function and surface expression were investigated using phospho-mimicking and phosphodeficient mutations, coexpression of constitutively active kinases and pharmacological kinase induction in a heterologous expression system. Our results suggest that Ser277 is involved in an initial phase of PKC-mediated down-regulation of SERT. The five identified sites can guide future studies of direct links between SERT phosphorylation and regulatory processes.
Department of Drug Design and Pharmacology, University of Copenhagen , Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Lena
L
Sørensen
Kristian
K
Strømgaard
Anders S
AS
Kristensen
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D053719:Tandem Mass Spectrometry
D050486:Serotonin Plasma Membrane Transport Proteins