Abstract
Identification of oncogene-mediated phosphorylation events is essential to understanding the molecular determinants responsible for cancer development and progression. Here, we identify KRAS-regulated phosphorylation events using label-free quantitation-based comparative phosphoproteomics analyses of immortalized human bronchial epithelial cells that express oncogenic KRAS as well as cells that do not. Further, we demonstrate integration of the identified phosphorylation events with the Pathway Interaction Database to infer KRAS-regulated pathways, which may have implications in KRAS-associated lung adenocarcinoma development. Taken together, our study provides an overview of the functional phosphoproteomics approach involving cell culture, preparation of whole cell lysates, trypsin digestion, phosphopeptide enrichment, mass spectrometry analyses, label-free quantitative analyses, and signaling pathway analyses to study KRAS-targeted events.
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References
Malumbres M, Barbacid M (2003) RAS oncogenes: the first 30 years. Nat Rev Cancer 3:459–465
Riely GJ, Marks J, Pao W (2009) KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc 6:201–205
Qian J, Niu J, Li M et al (2005) In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis. Cancer Res 65:5045–5053
Tchernitsa OI, Sers C, Zuber J et al (2004) Transcriptional basis of KRAS oncogene-mediated cellular transformation in ovarian epithelial cells. Oncogene 23:4536–4555
Sudhir PR, Chen CH, Pavana Kumari M et al (2012) Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells. Mol Cell Proteomics 11:901–915
Guha U, Chaerkady R, Marimuthu A et al (2008) Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci U S A 105:14112–14117
Sudhir P-R, Hsu C-L, Wang M-J et al (2011) Phosphoproteomics identifies oncogenic ras signaling targets and their involvement in lung adenocarcinomas. PLoS One 6:e20199
Beltran L, Cutillas PR (2012) Advances in phosphopeptide enrichment techniques for phosphoproteomics. Amino Acids 43:1009–1024
Ramirez RD, Sheridan S, Girard L et al (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64:9027–9034
Sato M, Vaughan MB, Girard L et al (2006) Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res 66:2116–2128
Tsai CF, Wang YT, Chen YR et al (2008) Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics. J Proteome Res 7:4058–4069
Tsou CC, Tsai CF, Tsui YH et al (2010) IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation. Mol Cell Proteomics 9:131–144
Acknowledgment
This work was supported by the grant to JYC from National Research Program on Genomic Medicine, Taiwan and the postdoctoral fellowship to PRS from the Academia Sinica, Taiwan.
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Sudhir, PR., Chen, JY. (2014). Functional Phosphoproteomics of Oncogenic KRAS Signaling. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_10
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DOI: https://doi.org/10.1007/978-1-62703-791-4_10
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-790-7
Online ISBN: 978-1-62703-791-4
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