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Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation
M Zohar1, H Teramoto, B Z Katz
1Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, Bethesda, Maryland 20892-4330, USA.
Abstract:
The small GTP-binding Rho proteins control a variety of biological activities, including organization of the actin cytoskeleton, regulation of gene expression and cellular transformation. In contrast, Ras proteins do not induce actin stress fibers, but potently transform cells which exhibit a morphology clearly distinct from that caused by activated forms of Rho. To investigate whether nuclear signaling and oncogenic potential of Rho are a consequence of its profound effect on cytoskeletal organization, we replaced each amino acid in the Rho effector loop with those of Ras, or replaced conserved residues with others known to result in differential signaling capability when introduced into Ras and Rac1. These Rho mutants did not gain the ability to induce the MAPK, JNK or p38 pathways but, surprisingly, all Rho effector loop mutants still continued to induce actin stress fiber formation. However, three of these Rho mutants, with substitutions of leucine-39, glutamic acid-39, or cysteine-42, lost the ability to stimulate gene transcription via the serum response factor (SRF) and failed to induce neoplastic transformation. Thus, these results indicate that cytoskeletal changes are not sufficient to induce the transformed phenotype, and that Rho-effector molecules regulating the actin cytostructure are distinct from those signaling to the nucleus and subverting normal growth control.
Insights
Rho proteins regulate cell functions, but their oncogenic potential isn't solely due to cytoskeletal changes. Specific mutations disrupt gene transcription and transformation, separating these cellular effects.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rho proteins are small GTP-binding proteins crucial for actin cytoskeleton organization, gene expression, and cellular transformation.
- Ras proteins, distinct from Rho, potently transform cells without inducing actin stress fibers.
Purpose of the Study:
- To investigate if Rho's nuclear signaling and oncogenic potential stem from its effects on cytoskeletal organization.
- To differentiate the molecular mechanisms underlying Rho-mediated cytoskeletal changes versus transformation.
Main Methods:
- Mutagenesis of the Rho effector loop by replacing amino acids with those from Ras or other variants.
- Analysis of Rho mutant signaling pathways, including MAPK, JNK, and p38.
- Assessment of actin stress fiber formation, gene transcription via serum response factor (SRF), and neoplastic transformation.
Main Results:
- Rho effector loop mutants retained the ability to induce actin stress fiber formation.
- Mutants with substitutions at leucine-39, glutamic acid-39, or cysteine-42 lost the capacity to stimulate SRF-mediated gene transcription.
- These specific Rho mutants also failed to induce neoplastic transformation, despite maintaining cytoskeletal effects.
Conclusions:
- Cytoskeletal organization changes induced by Rho are insufficient for neoplastic transformation.
- Rho-effector molecules responsible for actin cytostructure regulation are distinct from those mediating nuclear signaling and growth control subversion.
- This suggests a divergence in Rho signaling pathways controlling cellular morphology and oncogenic potential.