Structure/Function Analysis of a Bacterial Protein Kinase Phosphoprotein Phosphatase

超过20种不同的细菌适应性反应， 环境刺激由蛋白质家族调节， 反应调节因子，共有一个保守的氨基末端 域 这些反应调节剂的活性是 由第二个蛋白质家族控制， 结构域，通常位于羧基末端。 最近 用纯化的信号转导蛋白的实验 控制氮同化（NTR调节子），趋化性 (Che)和OMP调节子（OMP regulon）表明， 该第二家族的蛋白质是蛋白激酶， 在某些情况下，磷酸蛋白磷酸酶调节 适应性反应通过控制 反应调节剂的磷酸化。 这些蛋白 激酶，最好理解的是NRII，其调节氮 通过催化磷酸化和同化 增强子结合转录的去磷酸化 NRI因子。在这个提议中，PI提出了实验 旨在进一步阐明激酶的机制 NRII催化的磷酸酶反应的结果 这些实验可能适用于所有的 同源蛋白激酶。 NRII有三项活动： 在ATP的存在下，NRII在一个 组氨酸残基。 然后将磷酰基转移到 连接到N-末端结构域内的N-乙酰基残基， 响应调节器NRI。 最后，在在场的 信号转导蛋白PII和ATP，NRII催化 P-NRI的去磷酸化。 基因实验是 建议应规定核苷酸的要求 结合和自身激酶，磷酸转移酶。和 通过分离或构建突变体的磷酸酶活性 尤其是在这些活动中存在不足。 此外，本发明还提供了一种方法， 实验提出，应直接阐明 自磷酸化组氨酸残基的身份， 自磷酸化的化学计量，以及 核苷酸结合

Over 20 different bacterial adaptive responses to environmental stimuli are regulated by a family of proteins, response regulators, sharing a conserved amino terminal domain. The activity of these response regulators is controlled by a second family of proteins sharing a conserved domain, usually located at the carboxyl terminus. Recent experiments with the purified signal transduction proteins that control nitrogen assimilation (NTR regulon), chemotaxis (Che) and osmoregulation (OMP regulon) indicate that the proteins of this second family are protein kinases and, in some cases, phosphoprotein phosphatases that regulate the adaptive responses by controlling the level of phosphorylation of the response regulators. Of these protein kinases, the best understood is NRII which regulates nitrogen assimilation by catalyzing the phosphorylation and dephosphorylation of the enhancer-binding transcription factor NRI. In this proposal, the PI presents experiments designed to further elucidate the mechanisms of the kinase and phosphatase reactions catalyzed by NRII The results of these experiments will probably be applicable to all of the homologous protein kinases. NRII has three activities: in the presence of ATP, NRII is autophosphorylated on a histidine residue. This phosphoryl group is then transferred to an aspartyl residue within the N-terminal domain of the response regulator NRI. Finally, in the presence of the signal transduction protein PII and ATP, NRII catalyzes the dephosphorylation of P-NRI. Genetic experiments are proposed that should define the requirements for nucleotide binding and the autokinase, phosphotransferase. and phosphatase activities by isolating or constructing mutants specifically deficient in these activities. In addition, experiments are proposed that should directly elucidate the identity of the autophosphorylated histidine residue, the stoichiometry of autophosphorylation, and the sites of nucleotide binding.