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 inhibito r of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS-7 cells e xpressing recombinant serotonin transporter (SET). In BeWo cells, trea tment with TPA decreased imipramine-sensitive serotonin uptake with a reduction in V-max without affecting K-m. CLA also decreased imipramin e-sensitive serotonin uptake in a manner similar to that of TPA. TPA a nd 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 we re reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phos phorylation of SET by PKC, site-directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was a lso down-regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct ph osphorylation of SET by PKC.