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A novel Cdc42Hs mutant induces cellular transformation
1Department of Pharmacology, Cornell University, Ithaca, New York 14853-6401, USA.
Abstract:
Cdc42Hs is a small GTPase of the Rho-subfamily, which regulates signaling pathways that influence cell morphology and polarity, cell-cycle progression and transcription. An essential role for Cdc42Hs in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator-a guanine nucleotide exchange factor (GEF)-for Cdc42Hs, and that activated mutants of the closely related GTPases Rac and Rho are transforming. As we were unable to obtain significant over-expression of GTPase-defective Cdc42Hs mutants, we have generated a mutant, Cdc42Hs(F28L), which can undergo spontaneous GTP-GDP exchange while maintaining full GTPase activity, and thus should exhibit functional activities normally imparted by Dbl. In cultured fibroblasts, Cdc42Hs(F28L) activated the c-Jun kinase (JNK1) and stimulated filopodia formation. Cells stably expressing Cdc42Hs(F28L) also exhibited several hallmarks of transformation-reduced contact inhibition, lower dependence on serum for growth, and anchorage-independent growth. Our findings indicate that Cdc42Hs plays a role in cell proliferation, and is a likely physiological mediator of Dbl-induced transformation.
Insights
The Rho-subfamily GTPase Cdc42Hs regulates cell growth and morphology. A specific mutant, Cdc42Hs(F28L), promotes cell proliferation and transformation, suggesting its role in Dbl-induced oncogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Cdc42Hs, a Rho-subfamily GTPase, regulates critical cellular processes including morphology, polarity, cell cycle, and transcription.
- The Dbl oncoprotein, a guanine nucleotide exchange factor (GEF), activates Cdc42Hs, and related GTPases like Rac and Rho, when activated, are transforming.
- Previous attempts to over-express GTPase-defective Cdc42Hs mutants were unsuccessful.
Purpose of the Study:
- To investigate the role of Cdc42Hs in cell proliferation and transformation.
- To generate and characterize a novel Cdc42Hs mutant, Cdc42Hs(F28L), that exhibits spontaneous GTP-GDP exchange and retains GTPase activity, mimicking Dbl activation.
- To determine if Cdc42Hs(F28L) can induce cellular transformation phenotypes.
Main Methods:
- Generation of a constitutively active Cdc42Hs mutant (Cdc42Hs(F28L)).
- Expression of Cdc42Hs(F28L) in cultured fibroblasts.
- Assays to measure c-Jun kinase (JNK1) activation and filopodia formation.
- Assessment of transformation hallmarks: reduced contact inhibition, decreased serum dependence, and anchorage-independent growth.
Main Results:
- Cdc42Hs(F28L) expression activated JNK1 and stimulated filopodia formation in fibroblasts.
- Stable expression of Cdc42Hs(F28L) resulted in cells with reduced contact inhibition.
- Cells expressing Cdc42Hs(F28L) showed lower serum dependence for growth and exhibited anchorage-independent growth.
Conclusions:
- Cdc42Hs plays a significant role in regulating cell proliferation.
- The Cdc42Hs(F28L) mutant demonstrates functional similarities to Dbl-mediated activation.
- Cdc42Hs is implicated as a likely physiological mediator of Dbl-induced cellular transformation.