Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization*
- From the ‡Department of Biochemistry, Biophysics, and Molecular Biology, ¶the Department of Zoology/Genetics, and the §Signal Transduction Training Group, Iowa State University, Ames, Iowa 50011
Abstract
In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.
Footnotes
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↵* This work was supported by funds (to J.E.B.) from the Roy J. Carver Charitable Trust, the Elsa U. Pardee Foundation, and NSF Professional Opportunities for Women in Research and Education Award MCB 9973378.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: 3212 Molecular Biology Bldg., Iowa State University, Ames, IA 50011. Tel.: 515-294-6125; Fax: 515-294-0453; E-mail: jbuss@iastate.edu.
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Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M001368200
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↵2 M. A. Booden and J. E. Buss, unpublished results.
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↵3 M. A. Booden and J. E. Buss, unpublished data.
- Abbreviations:
- PI3-kinase
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phosphatidylinositol 3-kinase
- HA
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hemagglutinin
- 4-Ptase
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inositol 4-phosphatase
- DRM
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detergent-resistant membrane
- PAGE
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polyacrylamide gel electrophoresis
- MEK
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mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
- ERK
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extracellular signal-regulated kinase
- PAK
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p21-activated kinase
- PtdIns
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phosphatidylinositol
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- Received February 18, 2000.
- Revision received May 2, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
