Modulation of serotonin transporter activity by a protein kinase C activator and an inhibitor of type 1 and 2A serine/threonine phosphatases.
We studied the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and calyculin A (CLA), an inhibitor of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS-7 cells expressing recombinant serotonin transporter (SET). In BeWo cells, treatment with TPA decreased imipramine-sensitive serotonin uptake with a reduction in Vmax without affecting Km. CLA also decreased imipramine-sensitive serotonin uptake in a manner similar to that of TPA. TPA and CLA also decreased the uptake activity of recombinant SET expressed in COS-7 cells as seen in BeWo cells. These effects of TPA and CLA were reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phosphorylation of SET by PKC, site-directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was also down-regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct phosphorylation of SET by PKC.
Sakai
N
N
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-ku, Japan.
Sasaki
K
K
Nakashita
M
M
Honda
S
S
Ikegaki
N
N
Saito
N
N
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D019556:COS Cells / Q000737:chemistry / Q000201:enzymology; D002273:Carcinogens / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002822:Choriocarcinoma; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D008387:Marine Toxins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009419:Nerve Tissue Proteins; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011493:Protein Kinase C / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D012701:Serotonin / Q000378:metabolism / Q000493:pharmacokinetics; D050486:Serotonin Plasma Membrane Transport Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000201:enzymology
D018759:Adrenergic Uptake Inhibitors; D002273:Carcinogens; D002352:Carrier Proteins; D008387:Marine Toxins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010080:Oxazoles; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D014316:Tritium; D012701:Serotonin; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D013755:Tetradecanoylphorbol Acetate; D007099:Imipramine