Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase.
Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase.
复制标题
血清素转运的循环 GMP 依赖性刺激不涉及 cGMP 依赖性蛋白激酶的直接转运蛋白磷酸化。
DOI：
10.1074/jbc.m112.394726
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Rudnick,Gary
中科院分区：
文献类型：
--
作者：
Wong,Albert;Zhang,Yuan-Wei;Jeschke,GraceR;Turk,BenjaminE;Rudnick,Gary
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 mannerin 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 analogN6-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 SERTin vitroled to radiolabel incorporation from [γ-33P]ATP but not from similarly labeledN6-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.
