Abstract
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.
Highlights
The biochemical and behavioral effects of SERT modulation of SERT modulation using exogenous agents suggest that SERT expression may be tightly regulated in vivo and, perhaps, altered in its regulation in disease states [17]
We report the use of these antibodies to establish the direct phosphorylation of hSERT proteins using 293-hSERT cells. hSERT proteins in this system are phosphorylated under basal conditions, and phosphorylation can be significantly elevated by both PKC and cyclic nucleotide-activated protein kinases
Immunoprecipitation of Phosphorylated hSERT Protein—To determine whether hSERT proteins are subject to phosphorylation, we metabolically labeled stably transfected 293-hSERT cells with [32P]orthophosphate and immunoprecipitated detergent extracts with a set of SERT-specific and control antisera
Summary
Materials—293-hSERT cells were previously generated and characterized in this laboratory [1]. Cell Culture—293-hSERT and parental HEK-293 lines were maintained in monolayer culture in 75-cm flasks in an atmosphere of 5% CO2 at 37 °C as described previously [1] Both lines were grown in DMEM containing 10% dialyzed fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. The adherent cells were washed three times with phosphate-buffered saline and lysed by the addition of 400 l/well ice-cold modified radioimmunoprecipitation (RIPA, 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4) buffer containing protease (1 M pepstatin A, 250 M phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin) and phosphatase inhibitors (10 mM sodium fluoride, 50 mM sodium pyrophosphate, and 1 M okadaic acid) for 1 h at 4 °C with agitation. Quantitation from digitized autoradiograms was evaluated on multiple film exposures to ensure quantitation within the linear range of the film and gave identical results to estimations achieved with direct PhosphorImager quantitation
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