Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines.
Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.
Unidad de Investigación, Hospital Universitario de Salamanca, Spain.
Laura
L
Ferreira
Isabel
I
Fuentes-Calvo
José M
JM
Muñoz-Félix
Carmen
C
Muñiz-Martín
Fernando
F
Sánchez-Juanes
César
C
Raposo
José M
JM
González-Buitrago
José M
JM
López-Novoa
Carlos
C
Martínez-Salgado
D000818:Animals; D002461:Cell Line, Transformed; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005347:Fibroblasts; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D005838:Genotype; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000096:biosynthesis / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D020730:Guanine Nucleotide Dissociation Inhibitors; D020543:Proteome; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D007770:L-Lactate Dehydrogenase; D016283:Proto-Oncogene Proteins p21(ras)