Kamberov E S, Atkinson M R, Chandran P, Ninfa A J
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1994 Nov 11;269(45):28294-9.
We examined the effects of mutations in glnL, encoding the signal-transducing kinase/phosphatase nitrogen regulator II (NRII), on the regulated phosphatase activity involved in nitrogen regulation. With wild-type NRII, this phosphatase activity was only observed in the presence of the signal transduction protein II (PII). Three different glnL mutations result in altered NRII proteins that had phosphatase activity in the absence of PII. The most active of these contained an alteration of the site of NRII autophosphorylation, histidine 139, to asparagine (H139N). The phosphatase activity of the NRII-H139N protein was further stimulated by the PII protein and by ATP. This suggests that the PII protein is not directly involved in a catalytic step of the regulated phosphatase activity but rather plays a regulatory role. We also measured the effect on the regulated phosphatase activity of alterations at conserved residues in the kinase/phosphatase domain of NRII and the effect of deleting the non-conserved N-terminal domain of NRII. For this we used fusion proteins containing the Escherichia coli maltose-binding protein (MBP) linked to the protein of interest. A protein consisting of MBP linked to wild-type NRII was a less active kinase than was wild-type NRII but in the presence of PII had wild-type phosphatase activity. A protein consisting of MBP linked to just the C-terminal domain of wild-type NRII had kinase activity but lacked phosphatase activity. Alterations at the highly conserved residues Asp-287, Gly-289, and Gly-291 in NRII affected both activities. A fusion of MBP to the NRII-H139N protein lacked kinase activity but had phosphatase activity in the absence of PII. Thus, while the kinase and phosphatase activities of NRII could be genetically separated, some of the highly conserved residues in the C-terminal domain of NRII (Asp-287, Gly-289, Gly-291) are apparently important for both activities.
我们研究了编码信号转导激酶/磷酸酶氮调节因子II（NRII）的glnL基因突变对参与氮调节的磷酸酶活性调控的影响。对于野生型NRII，只有在信号转导蛋白II（PII）存在的情况下才能观察到这种磷酸酶活性。三种不同的glnL突变导致NRII蛋白发生改变，使其在没有PII的情况下也具有磷酸酶活性。其中活性最高的一种突变是将NRII自身磷酸化位点的组氨酸139替换为天冬酰胺（H139N）。NRII-H139N蛋白的磷酸酶活性受到PII蛋白和ATP的进一步刺激。这表明PII蛋白并非直接参与调控磷酸酶活性的催化步骤，而是发挥调节作用。我们还测定了NRII激酶/磷酸酶结构域中保守残基的改变对调控磷酸酶活性的影响，以及删除NRII非保守的N端结构域的影响。为此，我们使用了含有与目标蛋白相连的大肠杆菌麦芽糖结合蛋白（MBP）的融合蛋白。由MBP与野生型NRII相连组成的蛋白作为激酶的活性低于野生型NRII，但在PII存在的情况下具有野生型磷酸酶活性。仅由MBP与野生型NRII的C端结构域相连组成的蛋白具有激酶活性，但缺乏磷酸酶活性。NRII中高度保守的残基天冬氨酸-287、甘氨酸-289和甘氨酸-291的改变影响了这两种活性。MBP与NRII-H139N蛋白的融合蛋白缺乏激酶活性，但在没有PII的情况下具有磷酸酶活性。因此，虽然NRII的激酶和磷酸酶活性可以通过基因方式分离，但NRII C端结构域中的一些高度保守残基（天冬氨酸-287、甘氨酸-289、甘氨酸-291）显然对这两种活性都很重要。
