Ramamoorthy Sammanda, Samuvel Devadoss J, Buck Eric R, Rudnick Gary, Jayanthi Lankupalle D
Department of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, South Carolina 29425.
J Biol Chem. 2007 Apr 20;282(16):11639-47. doi: 10.1074/jbc.M611353200. Epub 2007 Feb 19.
Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.
细胞蛋白激酶、磷酸酶及其他与5-羟色胺转运体（SERT）相互作用的蛋白参与了多种调节SERT活性的信号传导机制。我们研究了蛋白激酶G（PKG）介导的SERT调节的分子机制及转运体磷酸化位点。用8-溴-cGMP处理大鼠中脑突触体可增加SERT活性，且PKG抑制剂可选择性地阻断这一增加。cGMP处理后，5-羟色胺（5-HT）转运的V(max)值增加。然而，表面生物素化研究显示PKG激活后SERT的表面丰度没有变化。(32)P代谢标记实验表明，在存在cGMP的情况下SERT磷酸化增加，而选择性抑制PKG可消除这种增加。磷酸氨基酸分析显示，cGMP刺激的天然SERT磷酸化仅发生在苏氨酸残基上。当将8-溴-cGMP添加到表达SERT的CHO-1细胞中时，可刺激5-HT转运和SERT磷酸化。将SERT的苏氨酸276突变为丙氨酸可完全消除cGMP介导的5-HT转运刺激和SERT磷酸化。尽管T276A突变对5-HT转运或SERT蛋白表达没有显著影响，但突变为天冬氨酸（T276D）可使5-HT摄取水平提高到野生型SERT表达细胞中cGMP刺激的5-HT摄取水平，且不再对cGMP敏感。这些发现首次鉴定了SERT中的一个磷酸化位点，并证明Thr-276的磷酸化是cGMP介导的SERT调节所必需的。它们还构成了首个证据，即在中枢神经系统中PKG激活通过一种不依赖于转运的机制刺激内源性SERT活性。
