Deora A A, Hajjar D P, Lander H M
Departments of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Biochemistry. 2000 Aug 15;39(32):9901-8. doi: 10.1021/bi992954b.
Nitric oxide (NO) and related species serve as cellular messengers in various physiological and pathological processes. The monomeric G protein, Ras, transduces multiple signaling pathways with varying biological responses. We have previously reported that NO triggers Ras activation and recruitment of an effector, phosphatidylinositol 3'-kinase (PI3K) and Ras-dependent activation of mitogen-activated protein (MAP) kinases which include extracellular signal regulated kinases (ERKs), c-Jun NH(2)-terminal kinase (JNK), and p38 MAP kinase. In this study, we further defined NO-activated Ras signaling pathways. We have identified Raf-1 as another effector recruited by NO-activated Ras in T lymphocytes. NO activation results in association of Ras and Raf-1 and is biologically significant, as we observe an NO-induced increase in Raf-1 kinase activity. Downstream to Raf-1 kinase lie MAP kinases and their subsequent downstream targets, transcription factors. We found that treatment of T lymphocytes with NO yielded phosphorylation of the transcription factor, Elk-1. This phoshorylation is dependent on NO binding to the cysteine 118 residue of Ras. By further delineating the pathway with pharmacological inhibitors, Elk-1 phosphorylation was also found to be dependent on PI3K and ERK. Moreover, NO triggered an increase in mRNA levels of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), which was ERK dependent. Thus, we have defined an NO-induced signaling pathway in T lymphocytes arising at the membrane where NO-activated Ras recruits Raf-1 and culminating in the nucleus where Elk-1 is phosphorylated and TNF-alpha messenger RNA is induced. This NO-activated Ras-mediated signaling pathway may play a critical role in Elk-1-induced transcriptional activation of T lymphocytes, host defense and inflammation.
一氧化氮（NO）及相关物质在各种生理和病理过程中充当细胞信使。单体G蛋白Ras可转导多种具有不同生物学反应的信号通路。我们之前曾报道，NO可触发Ras激活以及效应分子磷脂酰肌醇3'-激酶（PI3K）的募集，还能引发Ras依赖性的丝裂原活化蛋白（MAP）激酶激活，其中包括细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38 MAP激酶。在本研究中，我们进一步明确了NO激活的Ras信号通路。我们已确定Raf-1是NO激活的Ras在T淋巴细胞中募集的另一种效应分子。NO激活导致Ras与Raf-1结合，且具有生物学意义，因为我们观察到NO诱导Raf-1激酶活性增加。Raf-1激酶的下游是MAP激酶及其随后的下游靶点转录因子。我们发现用NO处理T淋巴细胞会使转录因子Elk-1发生磷酸化。这种磷酸化依赖于NO与Ras的半胱氨酸118残基结合。通过用药物抑制剂进一步描绘该通路，还发现Elk-1磷酸化也依赖于PI3K和ERK。此外，NO引发促炎细胞因子肿瘤坏死因子-α（TNF-α）的mRNA水平升高，这是ERK依赖性的。因此，我们确定了T淋巴细胞中由NO诱导的信号通路，该通路起始于细胞膜，在那里NO激活的Ras募集Raf-1，最终在细胞核中Elk-1被磷酸化且TNF-α信使RNA被诱导。这种由NO激活的Ras介导的信号通路可能在Elk-1诱导的T淋巴细胞转录激活、宿主防御和炎症中起关键作用。
