GM1 expression in H-ras-transformed NIH3T3 results in the suppression of cell proliferation inducing the partial transfer of activated H-ras from non-raft to raft fraction

c-H-ras is located in lipid/rafts and undergoes cholesterol dependent regulation. To analyze the regulatory effects of ganglioside GM1 on the proliferation of fibroblasts transformed with mutated ras gene, GM1 synthase cDNA was transfected into NIH3T3/H-ras cells containing mutation. In the transient expression system with EGFP-fused GM1 synthase, the ratio of BrdU-positive cells among EGFP-positive cells was compared between GM1+ transfectant cells and control cells, indicating that the transient GM1 expression suppresses cell proliferation. Then, established transfectant cells C21 and C34 expressed definite levels of GM1, and analyzed for the cell growth with the control cells D2 and D4 expressing no GM1. GM1+ cells showed reduced proliferation compared with controls. Phosphorylation levels of ERK1/2 after FCS treatment were examined, showing that those on the GM1+ transfectant cells increased slowly, while those in the controls rapidly reached the plateau. Fractionation of Triton X-100 extracts with sucrose density gradient ultra-centrifugation revealed that activated H-ras proteins from controls as well as NIH3T3/H-ras were completely localized in non-GEM/raft fraction. On the other hand, some portions of activated H-ras were transferred to GEM/raft fraction, i.e., 32% in C21, and 8% in C34. Since the Ras effector Raf-1 was localized in non-GEM/raft, the growth suppression might be caused, at least partly, by the movement of activated H-ras to GEM/raft fraction.