Abstract
The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including "ecstasy" (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N(6)-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-(33)P]ATP but not from similarly labeled N(6)-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.
Highlights
Serotonin transporter (SERT) phosphorylation and transport activation requires cyclic GMP-dependent protein kinase (PKG)
N6-Benzyl-ATP inhibited both M438A and M438G mutants, whereas wild type (WT) kinase was essentially insensitive at analog concentrations up to 800 M
The M438G mutant utilized N6-benzyl-ATP as a substrate relatively efficiently, with a Km value one-third of that measured for ATP and a Vmax slightly higher than the WT enzyme with ATP as a substrate (Table 1)
Summary
Serotonin transporter (SERT) phosphorylation and transport activation requires cyclic GMP-dependent protein kinase (PKG). In RBL cells, derived from 5-HT accumulating basophils, activation of the A3 adenosine receptor led to increased SERT activity [3] This activation was blocked by inhibitors of nitric oxide synthase (NOS) and cyclic GMP-dependent protein kinase (PKG), suggesting a pathway in which the A3 receptor stimulates Ca2ϩ entry, which in turn activates NOS, leading to synthesis of cGMP by soluble guanylyl cyclase. Cysteine-scanning mutagenesis of the region around Thr-276 indicated that this residue is relatively inaccessible compared with neighboring residues and that the region is ␣-helical, an unlikely conformation for phosphorylation by kinases, which typically bind substrates in an extended conformation [12] For this reason, we sought to test whether SERT was a direct substrate for PKG. We describe the generation of analogsensitive PKG I␣ and its use in testing direct phosphorylation of SERT by PKG
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