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[Inhibition of ras-dependent transformation by using dominant negative ras mutant N116Y]
1Department of Plastic and Reconstructive Surgery, Hokkadio University School of Medicine, Sapporo, Japan.
Abstract:
Ras p21s are known as molecular switch for signal transduction pathways. They act as intracellular signal transducers of extracellular signals for growth and differentiation. Ras activities are regulated by the rotation between active GTP-bound form and inactive GDP-bound form. This cycle is regulated by the GDP/GTP exchange reaction and intrinsic GTPase activity of ras p21. The N116Y, v-H-ras mutant substituted the asparagine-116 with tyrosine, has dominant negative activity toward normal ras p21, and suppresses ras dependent transformed phenotypes. To investigate the effects of N116Y on ras-mediated signals for transformation, I constructed an inducible vector by recombination of the N116Y mutant to the downstream of human metallothionein promoter, and transfected it into an NIH3T3 cell line transformed by LTR linked normal c-H-ras, 18A. I isolated two 18A cell clones T1 and T6. Both of the cell lines were able to induce the N116Y mutant after the heavy metal treatment. These clones displayed flat reversion within 24 hours. In addition, these clones also inhibited colony formation in soft agar by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or serum stimulation. The N116Y mutant blocked GDP/GTP exchange reaction by each growth stimulation. On the other hand, this mutant could not have sufficient influence upon extracellular signal-regulated kinase 2 (ERK2) phosphorylation, which located downstream of ras-mediated signal transduction, provoked by PDGF and serum stimulation. These results suggest that ERK2 activation is not necessary and sufficient for ras-dependent transformation. There could be a divergency in signal transduction between cell growth and transformation. The signal suppressed by the N116Y mutant may play an important role in cellular transformation.
Insights
The N116Y mutant suppresses Ras-dependent cell transformation by blocking GDP/GTP exchange. ERK2 activation is not essential for Ras-mediated transformation, suggesting distinct signaling pathways for cell growth and transformation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Context:
- Ras p21 proteins function as molecular switches in signal transduction pathways, regulating cell growth and differentiation.
- Ras activity is controlled by the cycling between active GTP-bound and inactive GDP-bound states, influenced by GDP/GTP exchange and GTPase activity.
- The v-H-ras N116Y mutant exhibits dominant-negative activity, suppressing Ras-dependent phenotypes.
Purpose:
- To investigate the impact of the N116Y mutant on Ras-mediated signals crucial for cellular transformation.
- To construct an inducible expression system for the N116Y mutant in Ras-transformed NIH3T3 cells.
- To elucidate the role of ERK2 phosphorylation in Ras-dependent transformation.
Summary:
- An inducible vector expressing the N116Y mutant was created and transfected into Ras-transformed NIH3T3 cells (18A), yielding clones T1 and T6.
- Treatment with heavy metals induced N116Y expression, causing flat reversion and inhibiting anchorage-independent growth stimulated by EGF, PDGF, or serum.
- The N116Y mutant blocked GDP/GTP exchange but had limited effect on PDGF/serum-induced ERK2 phosphorylation, indicating ERK2 is not sufficient for Ras transformation.
Impact:
- These findings suggest that ERK2 activation is neither necessary nor sufficient for Ras-dependent cellular transformation.
- The study highlights a potential divergence in signal transduction pathways governing cell growth versus transformation.
- The N116Y-suppressed signal pathway may be critical for driving cellular transformation, offering new therapeutic targets.