Background Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. other novel cascades, in governing diverse functions such as gene expression, 414864-00-9 IC50 apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. 414864-00-9 IC50 Conclusions/Significance This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer. Introduction Constitutive activation of Ras-mediated signaling and its downstream components, such as MAP family kinases, play an important role in the pathogenesis of human cancers 1,2]. Lung cancer represents a good choice for investigating the molecular mechanisms of Ras-mediated MAPK signaling, because the activation of Ras oncogenes by mutation or amplification has been reported most frequently in lung cancer 1,3]. Among the post-translational modifications, reversible protein phosphorylation is a dominant regulatory mechanism involved in the oncogenic signaling process. Simultaneous identification and quantification of phosphorylation events induced by oncogenic signaling not only would provide insight into signaling mechanisms but also are essential for understanding the molecular determinants of disease progression. Despite decades of intensive analysis of the Ras family of proto-oncogenes (and and incorporation of stable isotopes into samples has been introduced and is extensively used 6,7]. However, the isotopic labeling-based quantitation limits the analysis of a large number of samples 414864-00-9 IC50 in a single experiment. Hence, label-free quantitation methods have gained more popularity in recent years 8]. To gain further information on Ras-regulated cellular processes, we conducted a pathway-based investigation to evaluate Ras activity. We identified Ras-mediated phosphorylation events in immortalized human bronchial epithelial cells (HBECs) using IDEAL-Q (ID-based elution time prediction by fragmental regression)-based quantitation proteomics, followed by computational methods to infer Ras-mediated signaling pathways and molecular functions. Furthermore, the interpretation of Ras-mediated phosphorylation targets and related pathways allowed us to demonstrate the involvement of Ras-mediated signaling in lung 414864-00-9 IC50 adenocarcinoma (AD), but not in large cell carcinoma (LCC). Our findings on the phosphorylation events, kinomes, and pathways regulated by oncogenic Ras and differential activation of Ras downstream signaling in lung AD and LCC could serve as a basis for future investigations elucidating the molecular mechanisms involved in the pathogenesis of human cancers. Methods Cell culture Cdk4 (cyclin-dependent kinase 4)/hTERT (human telomerase reverse transcriptase)-immortalized human bronchial epithelial cells (HBEC3-KT or 3KT) and K-RASV12-transformed HBEC3-KT cells (3KTR) were maintained in K-CFM medium containing Rabbit polyclonal to KLF8 50 g/mL bovine pituitary extract (BPE) and 5 ng/mL EGF under 5% CO2 and 37C, as previously described 9,10]. These cells were the kind gift of Dr. John D. Minna (University of Southwestern Medical Center, Dallas, TX, USA). The human non-small-cell lung cancer (NSCLC) H1299, H322, and A549 cell lines were purchased from the American Type Culture Collection. H1299 and H322 cells were grown in RPMI medium supplemented with 10% fetal bovine serum and 1% antibiotics under tissue culture conditions. A549 cells were grown under similar conditions in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics. Knockdown experiments, lysis, western blot analysis, tryptic digestion, and phosphopeptide enrichment The lentivirus-based knockdown approach has been previously 414864-00-9 IC50 described [11]. The pLKO.1-short hairpin RNA (shRNA) plasmids encoding an shRNAs with sequences targeting the firefly and with sequences targeting human KRAS (for 5 min, the cell lysates were collected. Western blotting was performed as previously described [11]. Tube-gel digestion was performed as previously described [12] with modifications. Phosphopeptides were purified using a revised one-step IMAC as previously described [13]. For more details, see Methods S1. Mass spectrometry analysis and label-free quantitation Purified phosphopeptide samples were desalted and analyzed by LC-MS/MS (Waters.