Balasubramanian N, Levay K, Keren-Raifman T, Faurobert E, Slepak V Z
Department of Molecular and Cellular Pharmacology and Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
Biochemistry. 2001 Oct 23;40(42):12619-27. doi: 10.1021/bi015624b.
In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser(427) and Ser(428). Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca(2+) ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.
在脊椎动物光感受器中，光激发的视紫红质与G蛋白转导素相互作用，使其结合GTP并刺激酶cGMP磷酸二酯酶。该信号通路活性状态的快速终止依赖于一种光感受器特异性的G蛋白信号调节因子RGS9-1，它作为转导素的GTP酶激活蛋白（GAP）发挥作用。在此，我们表明，在光感受器外段（OS）制剂中，RGS9-1很容易被一种内源性丝氨酸/苏氨酸蛋白激酶磷酸化。蛋白激酶C和丝裂原活化蛋白激酶抑制剂使标记减少约30%，而细胞周期蛋白依赖性激酶5（CDK5）和钙/钙调蛋白依赖性蛋白激酶II（CaMK II）抑制剂则无作用。环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）抑制剂H89使RGS9-1标记减少超过90%，而二丁酰环磷腺苷（dibutyryl-cAMP）则使其增加3倍，这表明PKA是负责OS中RGS9-1磷酸化的主要激酶。RGS9-1属于一个RGS亚家族，该家族还包括RGS6、RGS7和RGS11，它们与G蛋白β亚基Gβ5以异二聚体形式存在。磷酸化的RGS9-1仍与Gβ5L相关联，Gβ5L是一种光感受器特异性剪接形式，其本身未被磷酸化。从OS免疫沉淀的RGS9-1可被外源性PKA在体外磷酸化。PKA催化亚基也可使重组RGS9-1磷酸化，突变分析将磷酸化位点定位到丝氨酸（Ser）427和丝氨酸428。将这些残基替换为谷氨酸以模拟磷酸化，导致RGS9-1的GAP活性降低。在OS中，RGS9-1磷酸化需要游离钙离子的存在，并受到光的抑制，这表明RGS9-1磷酸化可能是介导暗适应细胞中更强光反应的机制之一。
