Recruitment and activation of Raf-1 kinase by nitric oxide-activated Ras.
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.
Departments of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
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D000730:Androstadienes / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000077191:Wortmannin; D050807:ets-Domain Protein Elk-1; D018631:ras Proteins / Q000378:metabolism*
D000730:Androstadienes; D002867:Chromones; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D050807:ets-Domain Protein Elk-1; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D000077191:Wortmannin