Phosphorylation and regulation of antidepressant-sensitive serotonin transporters.
Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) are responsible for efficient synaptic clearance of extracellular 5HT. Previously (Qian, Y., Galli, A., Ramamoorthy, S., Risso, S., DeFelice, L. J., and Blakely, R. D. (1997) J. Neurosci. 17, 45-47), we demonstrated that protein kinase (PKC)-linked pathways in transfected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the present study, we report that PKC activators rapidly, and in a concentration-dependent manner, elevate the basal level of hSERT phosphorylation 5-6-fold. Similarly, protein phosphatase (PP1/PP2A) inhibitors down-regulate 5HT transport and significantly elevate hSERT 32P incorporation, effects that are additive with those of PKC activators. Moreover, hSERT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindolylmaleimide I, whereas hSERT phosphorylation induced by phosphatase inhibitors is insensitive to these agents at comparable concentrations. Protein kinase A and protein kinase G activators fail to acutely down-regulate 5HT uptake but significantly enhance hSERT phosphorylation. Basal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent protein kinase inhibitors. Together these results reveal hSERT to be a phosphoprotein whose phosphorylation state is likely to be tightly controlled by multiple kinase and phosphatase pathways that may also influence the transporter's regulated trafficking.
Ramamoorthy
S
S
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.
Giovanetti
E
E
Qian
Y
Y
Blakely
R D
RD
D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D015290:Second Messenger Systems; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D013237:Stereoisomerism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D019319:Okadaic Acid; D012701:Serotonin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate