PHOSPHORYLATION AND REGULATION OF ANTIDEPRESSANT-SENSITIVE SEROTONIN TRANSPORTERS

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transpor ters (SERTs) are responsible for efficient synaptic clearance of extra cellular 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 transf ected HEK-293 cells lead to the internalization of cell-surface human (h) SERT protein and a reduction in 5HT uptake capacity. In the presen t 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-regu late 5HT transport and significantly elevate hSERT P-32 incorporation, effects that are additive with those of PKC activators. Moreover, hSE RT phosphorylation induced by beta-phorbol 12-myristate 13-acetate is abolished selectively by the PKC inhibitors staurosporine and bisindol ymaleimide I, whereas hSERT phosphorylation induced by phosphatase inh ibitors is insensitive to these agents at comparable concentrations. P rotein kinase A and protein kinase G activators fail to acutely down-r egulate 5HT uptake but significantly enhance hSERT phosphorylation. Ba sal hSERT and okadaic acid-induced phosphorylation were insensitive to chelation of intracellular calcium and Ca2+/calmodulin-dependent prot ein kinase inhibitors. Together these results reveal hSERT to be a pho sphoprotein whose phosphorylation state is likely to be tightly contro lled by multiple kinase and phosphatase pathways that may also influen ce the transporter's regulated trafficking.