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Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1
1First Department of Pathology and First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan.
Abstract:
The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.
Insights
The nm23H1 protein negatively regulates Rac1 activation by interacting with Tiam1, a Rac1-specific nucleotide exchange factor. This regulation of Rac1 by nm23H1 may contribute to its role as a tumor metastasis suppressor.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The nm23H1 protein, identified as a tumor metastasis suppressor, possesses nucleoside diphosphate kinase activity and influences cellular growth.
- Previous research indicated nm23H1 acts as a GTPase-activating protein for Rad, a Ras-related GTPase.
- The study investigated whether nm23H1 also regulates Rho-family GTPases, a key group of signaling proteins.
Purpose of the Study:
- To determine if nm23H1 regulates Rho-family GTPases.
- To elucidate the mechanism by which nm23H1 might influence Rac1 activation.
- To explore the functional consequences of nm23H1's interaction with Rac1 regulatory pathways.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- Western blotting to assess GTP-bound GTPase levels.
- Cell-based assays to evaluate Rac1 activation and downstream signaling (c-Jun kinase).
- Overexpression studies in Rat1 fibroblasts to observe effects on cell morphology (membrane ruffles).
Main Results:
- nm23H1 associated with Tiam1, a Rac1-specific nucleotide exchange factor, but not directly with Rho-family GTPases.
- Overexpression of nm23H1 inhibited Tiam1-induced Rac1 activation and c-Jun kinase activity.
- nm23H1's nucleoside diphosphate kinase activity converted GDP-bound Rac1, Cdc42, and RhoA to GTP-bound forms in vitro.
- nm23H1 overexpression reduced adhesion-stimulated membrane ruffles in Rat1 fibroblasts.
Conclusions:
- nm23H1 negatively regulates Tiam1, thereby inhibiting Rac1 activation in vivo.
- nm23H1's nucleoside diphosphate kinase activity plays a role in GTPase regulation.
- The identified function of nm23H1 in regulating Rac1 may be linked to its role as a tumor metastasis suppressor.