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Mutation of Ha-Ras C terminus changes effector pathway utilization
M A Booden1, D S Sakaguchi, J E Buss
1Department of Biochemistry, Biophysics, and Molecular Biology, the Department of Zoology/Genetics, and the Signal Transduction Training Group, Iowa State University, Ames, Iowa 50011, USA.
The Journal of Biological Chemistry
|May 10, 2000
Summary
A modified Ha-Ras protein (Ext61L), lacking farnesyl, enhances neuronal differentiation by altering actin cytoskeleton and lamellipodia formation. This suggests new ways to influence Ha-Ras effector utilization through C-terminal lipid modifications.
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- Ha-Ras protein localization to the plasma membrane is crucial for neuronal differentiation.
- Plasma membrane localization typically requires farnesyl and palmitate lipid attachments to the C-terminus of Ha-Ras.
Purpose of the Study:
- To investigate the role of C-terminal lipid modifications on Ha-Ras function in neuronal differentiation.
- To compare the effects of a farnesyl-lacking, palmitoylated Ha-Ras mutant (Ext61L) with wild-type Ha-Ras.
Main Methods:
- PC12 cells were used to study neuronal differentiation.
- Expression of modified Ha-Ras proteins (Ext61L and Ha-Ras61L) and inhibitory proteins (Rho, Cdc42, Rac, p21-activated kinase).
- Assays for Rac GTP binding, phosphatidylinositol 3-kinase activity, B-Raf kinase activity, and ERK phosphorylation.
Main Results:
- Ext61L induced greater neurite outgrowth, actin cytoskeletal changes, and lamellipodia formation compared to Ha-Ras61L.
- Ext61L showed significantly increased Rac GTP binding and phosphatidylinositol 3-kinase activity.
- Ext61L exhibited reduced B-Raf kinase activation and ERK phosphorylation compared to Ha-Ras61L.
Conclusions:
- Accentuating phosphatidylinositol 3-kinase activation and suboptimally activating B-Raf kinase leads to enhanced neuronal differentiation.
- C-terminal alternations in Ha-Ras, specifically lipid modifications, represent a novel mechanism for influencing Ha-Ras effector utilization.
- The lipidated C-terminus plays a broader role in Ha-Ras biological functions than previously understood.