Proteomics Analysis Of H-Ras-Mediated Transformated NIH3T3 Cell

The Ras gene product is a monomeric membrane-localized G protein of 21 kd that functions as a molecular switch linking receptor and nonreceptor tyrosine kinase activation to downstream cytoplasmic or nuclear events. In addition to R-ras and M-ras genes, there are three potentially oncogenic ras genes in human cells, which encode four highly related proteins H-ras, N-ras and K-ras(K-rasA and K-rasB).Ras is a membrane-bound guanosine triphosphate (GTP)/guanosine diphosphate (GDP)-binding (G) protein that serves as a "molecular switch, " converting signals from the cell membrane to the nucleus. These chemical signals lead to protein synthesis and regulation of cell survival, proliferation, and differentiation. However, unlike the classic heterotrimeric G proteins,Ras exists as a monomer. There is a close structural and sequence homology between the monomeric Ras family G proteins andα-subunits of the classic trimeric G proteins Each Ras protein consists of about 190-ami no-acid residues that are highly conserved in the N and C termini. Most of the differences between these proteins occur in the near Oterminal hypervariable domain of about 25 amino acids, which is presumed to be responsible for their different functions.Cell proliferation, differentiation, and survival are regulated by a number of extracellular hormones, growth factors, and cytokines in complex organisms. These molecules serve as ligands for cellular receptors and communicate with the nucleus of the cell through a network of intracellular signaling pathways. In cancer cells, dysregulated cell signaling and proliferation may occur through overexpression or mutation of proto-oncogenes. One such proto-oncogene is ras, which functions as a molecular switch in a large network of signaling pathways, mainly controlling the differentiation or proliferation of cells. Each mammalian cell contains at least three distinct ras proto-oncogenes encoding closely related, but distinct proteins. Activating mutations in these Ras proteins result in constitutive signaling, thereby stimulating cell proliferation and inhibiting apoptosis.Oncogenic mutations in the ras gene are present in approximately 30% of all human cancers. K-ras mutations occur frequently in non-small-cell lung, colorectal, and pancreatic carcinomas;H-ras mutations are common in bladder, kidney, and thyroid carcinomas; N-ras mutations are found in melanoma, hepatocellular carcinoma, and hematologic malignancies. The ras-signaling pathway has attracted considerable attention as a target for anticancer therapy because of its important role in carcinogenesis.Although multiple mediators of Ras signaling have been discovered, the exact mechanism by which oncogenic Ras acts in the multistep process of oncogenesis is not completely understood, and many downstream functions of Ras signaling in cellular transformation remain a mystery. The molecular mechanism of Ras-mediated oncogenic transformation as a whole has been detected by cDNA expression array. Nevertheless, it should be stressed that mRNA levels do not necessarily correlate with protein levels. Recently, technological advances in proteomics allow us to examine the expression profiles at the protein level on a genomewide scale. During our research on the proteomics analysis, we noticed a recent report on proteomics analysis of H-Ras-mediated transformation in human ovarian cancer model. However, our proteomics analysis revealed different alterations in the cellular proteome of NIH3T3 cells transformed by H-Ras.We implemented a proteomics approach to the systematical analysis of the alterations in the proteome of NIH3T3 cells transformed by oncogenic H-RasV12. Forty-four proteins associated with Ras-mediated transformation have been identified, and 28 proteins were not previously reported. Most of these identified proteins, affected by Ras-mediated transformation, could be classified into several general groups according to their cellular functions, including signal transduction, cytoskeleton, metabolism, protein degradation, transcription, translation, and ionic homeostasis.With the accomplishment of the human genome project, the focus in biological science has transferred from genomic study to the proteomic study. The emergence of proteomics allows us tostudy the moclecular features on a large scale for the first time. The main technology in proteome study includes 2-dimensional electrophoresis used to separate the proteins in the cells and mass spectromrtry (MALDI/TOF/MS and ESI/Q/TOF/MS) to identify the proteins.Interestingly, several of the identified protein targets were components of the UP system, indicating the UP system was obviously affected by Rasactivated transformation. Moreover, a principal finding in our study was the up-regulation of gankyrin in Ras-transformed cells. This was the first evidence to show that oncoprotein gankyrin was implicated in Ras-activated transformation.