Analysis of k-ras nuclear expression in fibroblasts and mesangial cells

Abstract

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.

Methodology/principal findings: 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/significance: 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.

Figure 1. Immunofluorescence analysis of cellular distribution of Ras isoforms in human mesangial cells.

Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression in human mesangial cells. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.

Figure 2. Immunofluorescence analysis of cellular distribution of Ras isoforms in H-ras ^−/−/N-ras ^−/− fibroblasts.

Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression in fibroblasts. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.

Figure 3. Genotypic and protein characterization of Hras ^−/−,Nras ^−/− and Kras ^−/− fibroblasts by PCR and Western blot.

(A) DNA PCR of K-ras ^−/− fibroblasts cultures; K-ras ^−/− fibroblasts are identified by a 270-bp product, and wild type fibroblasts by a 360-bp product (see methods section); mRNA (B) and protein (C) expression of Ras isoforms in K-ras ^−/− fibroblasts cultures; mRNA expression of H, N, K-Ras4A and K-Ras4B isoforms, evaluated by PCR (D), and protein expression (E) of H, N, and K-Ras isoforms, evaluated by Western blot, in Hras ^−/−,Nras ^−/− fibroblasts. K-Ras (1): mouse anti-human K-Ras (H3845-M01) from Abnova; K-Ras (2): mouse anti-mouse K-Ras (R3400) from Sigma.

Figure 4. Immunofluorescence analysis of cellular distribution of Ras isoforms in K-ras ^+/+ fibroblasts.

Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression) in K-ras ^+/+ fibroblasts. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.

Figure 5. Immunofluorescence analysis of cellular distribution of Ras isoforms in K-ras ^−/− fibroblasts.

Pictures show H-Ras, N-Ras, K-Ras4A and K-Ras4B expression) in K-ras ^−/− fibroblasts. The rhodamine red-labeled proteins are Ras isoforms, while the blue-labeled staining is Hoechst-stained DNA. Magnification: 630×. Scale bar: 30 µm. Antibodies: H-Ras, sc-520; K-Ras4A, sc-522; K-Ras4B, sc-521; N-Ras, sc-519.

Figure 6. K-Ras expression analysis in nuclear and cytoplasmic fractions of human mesangial cells and K-ras ^−/− fibroblasts by western blot.

K-Ras (1): mouse anti-human K-Ras (H3845-M01) from Abnova; K-Ras (2): mouse anti-mouse K-Ras (R3400) from Sigma; K-Ras (3): mouse anti-human K-Ras (sc-522) from Santa Cruz Biotechnologies. Cyclin A, histone H1 and tubulin were used as purity controls of nuclear (cyclin A, histone H1) and cytoplasmic fractions (tubulin), respectively.