Abstract
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.
MeSH terms
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Adrenergic Uptake Inhibitors / pharmacology
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Animals
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COS Cells / chemistry
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COS Cells / enzymology
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Carcinogens / pharmacology
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Choriocarcinoma
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Humans
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Imipramine / pharmacology
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Marine Toxins
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Membrane Transport Proteins*
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Mutagenesis, Site-Directed / physiology
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Nerve Tissue Proteins*
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Oxazoles / pharmacology
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Phosphoprotein Phosphatases / antagonists & inhibitors*
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Protein Kinase C / metabolism*
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Protein Phosphatase 1
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Rats
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Serotonin / metabolism
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Serotonin / pharmacokinetics
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Serotonin Plasma Membrane Transport Proteins
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Tetradecanoylphorbol Acetate / pharmacology
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Time Factors
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Transfection
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Tritium
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Tumor Cells, Cultured / chemistry
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Tumor Cells, Cultured / enzymology
Substances
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Adrenergic Uptake Inhibitors
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Carcinogens
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Carrier Proteins
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Marine Toxins
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Membrane Glycoproteins
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Membrane Transport Proteins
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Nerve Tissue Proteins
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Oxazoles
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SLC6A4 protein, human
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Serotonin Plasma Membrane Transport Proteins
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Slc6a4 protein, rat
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Tritium
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Serotonin
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calyculin A
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Protein Kinase C
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Phosphoprotein Phosphatases
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Protein Phosphatase 1
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Tetradecanoylphorbol Acetate
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Imipramine