calyculin-a and Choriocarcinoma

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.

Topics: Adrenergic Uptake Inhibitors; Animals; Carcinogens; Carrier Proteins; Choriocarcinoma; COS Cells; Humans; Imipramine; Marine Toxins; Membrane Glycoproteins; Membrane Transport Proteins; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase C; Protein Phosphatase 1; Rats; Serotonin; Serotonin Plasma Membrane Transport Proteins; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tritium; Tumor Cells, Cultured