Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments
Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments
复制标题
DOI：
10.1074/jbc.m011539200
复制
发表时间：
2001-06-22
影响因子：
4.8
通讯作者：
Palczewski, K
中科院分区：
文献类型：
--
作者：
Hu, G;Jang, GF;Palczewski, K
在从光激发恢复期间，视觉Gr蛋白转导素的失活受RGS 9 -1（普遍存在的RGS蛋白家族的GTP酶加速蛋白）调节。暗适应牛视杆外节与[γ-P-32]ATP孵育导致RGS 9 -1通过视杆外节膜中的内源性激酶磷酸化，平均化学计量为0.2-0.45 mol磷酸盐/mol RGS 9 -1。质谱分析揭示了一个主要的磷酸化位点，Ser（475）。负责催化重组RGS 9 -1而不是S（475）A突变体的强烈磷酸化的激酶。与Ser（475）周围区域对应的合成肽也被磷酸化，并且具有S（475）A取代的类似肽抑制RGS 9 -1磷酸化。RGS 9 -1激酶是在蔗糖梯度中与视紫红质共纯化的外周膜蛋白，并且可以在高离子强度的缓冲液中提取。它不被一组蛋白激酶A、蛋白激酶G、视紫红质激酶、CaM激酶II、酪蛋白激酶II或细胞周期蛋白依赖性激酶5的抑制剂或激活剂显著抑制或激活，浓度比其报道的IC 50或Ki值高50倍或更多倍。它被蛋白激酶C抑制剂双吲哚马来酰亚胺I和通过用EGTA将Ca 2+降低至纳摩尔水平来抑制，然而，在显著增强视紫红质磷酸化的条件下，它不被添加佛波酯刺激。对RGS 9 -1的Ser 475磷酸化形式特异的单克隆抗体在暗适应小鼠视网膜的免疫印迹中识别RGS 9 -1。来自光适应小鼠的视网膜具有低得多的RGS 9 -1磷酸化水平。因此，RGS 9 -1在体内在Ser（475）上被磷酸化，并且磷酸化水平受光和[Ca 2 +]调节，表明修饰在光适应中的重要性。
Inactivation of the visual Gr protein transducin, during recovery from photoexcitation, is regulated by RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS protein family. Incubation of dark-adapted bovine rod outer segments with [gamma-P-32]ATP led to RGS9-1 phosphorylation by an endogenous kinase in rod outer segment membranes, with an average stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass spectrometry revealed a single major site of phosphorylation, Ser(475). The kinase responsible catalyzed robust phosphorylation of recombinant RGS9-1 and not of an S(475)A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S(475)A substitution inhibited RGS9-1 phosphorylation. The RGS9-1 kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of inhibitors or activators of protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin dependent kinase 5, at concentrations 50 or more times higher than their reported IC50 or K-i values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca2+ to nanomolar levels with EGTA however, it was not stimulated by the addition of phorbol ester, under conditions that significantly enhanced rhodopsin phosphorylation, A monoclonal antibody specific for the Ser475 phosphorylated form of RGS9-1 recognized RGS9-1 in immunoblots of dark-adapted mouse retina. Retinas from light-adapted mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is phosphorylated on Ser(475) in vivo, and the phosphorylation level is regulated by light and by [Ca2+], suggesting the importance of the modification in light adaptation.