REGULATION OF SIGNALING BY G PROTEIN-BINDING PROTEINS

跨膜信号通常需要细胞表面受体、G蛋白和效应酶。RGS蛋白通过加速GTP水解和G蛋白失活来调节信号。信号也可以通过翻译后蛋白质修饰来调节。这一提议将检验RGS和G蛋白也受到翻译后调控的假设。这些实验将在酵母S.cerevisiae中进行，它表达一种与人类相似的G蛋白信号装置。酵母系统的使用将使体外生化方法和体内遗传策略能够以协调的方式使用，以解决以下四个目标：目标1：RGS蛋白是如何调节的？在此之前，我们用质谱仪证明了酵母中的RGS(Sst2)在体内是在Ser-380和Ser-539处被磷酸化的。Ser-539在信息素刺激下被磷酸化，需要一种MAP激酶，并稳定蛋白质。Ser-380的磷酸化还没有被鉴定。表达阵列文库将被用来鉴定使Ser-380磷酸化的激酶。然后，我们将使用激酶干扰和磷酸化位点突变体来确定这种修饰在体外如何改变G蛋白的催化活性，以及在体内如何改变G蛋白的信号活性。目的2：G蛋白是如何被调节的？酵母中的Galpha(Gpa1)已被证实是泛素化的，但该修饰的位置和功能尚不清楚。将使用质谱仪来鉴定泛素化的氨基酸。泛素化和蛋白降解途径中的突变将被用来确定G蛋白的降解机制。最后，基因中断和泛素化位点突变将被用来确定这一过程如何改变G蛋白的活性。目的3：G蛋白B亚单位是如何调节的？众所周知，酵母中的Gbeta(Ste4)是在信息素刺激下被磷酸化的。有间接证据表明，Ste4-磷酸化有助于信号脱敏。质谱仪将被用来确定磷酸化的位置。表达阵列文库将用于鉴定Ste4-Kinase。然后，我们将使用激酶干扰和磷酸化位点突变来确定这种修饰如何在体外和体内改变G蛋白的活性。目的4：调节RGS和G蛋白的其他修饰有哪些？随着质谱学的最新进展，现在应该可以识别感兴趣的蛋白质的所有修饰。RGS和G蛋白经历额外的(尚未确定的)修饰的可能性将被测试。每个修饰的分子和细胞后果将使用RGS和G蛋白功能的标准分析进行评估。

Transmembrane signaling typically requires a cell surface receptor, a G protein, and an effector enzyme. RGS proteins modulate signaling, by accelerating GTP hydrolysis and G protein inactivation. Signaling can also be modulated through post- translational protein modifications. This proposal will test the hypothesis that RGS and G proteins are also regulated post- translationally. These experiments will be carried out in the yeast S.cerevisiae, which expresses a G protein signaling apparatus similar to those in humans. The use of the yeast system will allow in vitro biochemical methods and in vivo genetic strategies to be used in a coordinated manner, to address the following four aims: Aim 1: How is the RGS protein regulated? Previously we used mass spectrometry to show that the RGS in yeast (Sst2) is phosphorylated at Ser-380 and Ser-539 in vivo. Ser-539 is phosphorylated in response to pheromone stimulation, requires a MAP kinase, and stabilizes the protein. Ser-380 phosphorylation has not been characterized. An expression array library will be used to identify the kinase that phosphorylates Ser-380. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein catalytic activity in vitro, and G protein signaling activity in vivo. Aim 2: How is the G protein a subunit regulated? It is established that the Galpha in yeast (Gpa1) is ubiquitinated, but the location and functional role of this modification are unknown. Mass spectrometry will be used to identify the ubiquitinated amino acid. Mutants in the ubiquitination and proteolysis pathways will be used to determine the mechanism of G protein degradation. Finally, gene disruption and ubiquitination-site mutants will be used to determine how this process alters G protein activity. Aim 3: How is the G protein B subunit regulated? It is known that the Gbeta in yeast (Ste4) is phosphorylated in response to pheromone stimulation. There is indirect evidence that Ste4-phosphorylation contributes to signal desensitization. Mass spectrometry will be used to identify the site of phosphorylation. The expression array library will be used to identify the Ste4-kinase. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein activity in vitro and in vivo. Aim 4: What other modifications regulate RGS and G proteins? With recent advances in mass spectrometry, it should now be possible to identify all modifications of a protein of interest. The likelihood that the RGS and G protein undergo additional (as yet unidentified) modifications will be tested. The molecular and cellular consequences of each modification will be evaluated using standard assays of RGS and G protein function.