Analysis of k-ras nuclear expression in fibroblasts and mesangial cells.
BACKGROUND
Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells.


RESULTS
Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras(-/-),N-ras(-/-) mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras(-/-)) embrionary fibroblasts by mating of K-ras(+/-) heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras(-/-) fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras(-/-) fibroblasts.


CONCLUSIONS
We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations.
Fuentes-Calvo
Isabel
I
Unidad de Fisiopatología Renal y Cardiovascular, Instituto Reina Sofía de Investigación Nefrológica, Universidad de Salamanca, Salamanca, Spain.
Blázquez-Medela
Ana M
AM
Santos
Eugenio
E
López-Novoa
José M
JM
Martínez-Salgado
Carlos
C
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D011905:Genes, ras; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016133:Polymerase Chain Reaction
D017931:DNA Primers