Related Experiment Videos
The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in
M Pomerance1, M N Thang, B Tocque
1Unité de Recherche sur la Glande Thyroïde et la Régulation Hormonale, U96 Institut National de la Santé et de la Recherche Médicale, Le Kremlin-Bicêtre, France.
Abstract:
The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.
Insights
Ras-GTPase-activating protein (RasGAP) SH3 domain is crucial for oncogenic Ras signaling in Xenopus oocytes. Blocking this domain prevents maturation-promoting factor activation, highlighting its role in cell cycle regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Ras-GTPase-activating protein (RasGAP) regulates p21ras-dependent signal transduction.
- The SH3 domain of RasGAP plays a role in this signaling pathway.
Purpose of the Study:
- Investigate the role of the RasGAP SH3 domain in signal transduction.
- Determine the involvement of RasGAP and MAP kinase pathways in Cdc2 activation.
Main Methods:
- Monoclonal antibody against RasGAP SH3 domain injection.
- Peptide inhibition assays targeting the SH3 domain.
- Expression of dominant-negative MAP kinase mutant protein (ERK2 K52R).
Main Results:
- Antibody against RasGAP SH3 domain specifically blocked oncogenic Ras-induced Cdc2 activation.
- SH3 domain peptides differentially affected Cdc2 activation.
- MAP kinase pathway is implicated in Cdc2 activation, independent of RasGAP SH3 domain's direct role.
Conclusions:
- Oncogenic Ras activates two distinct signaling pathways: MAP kinase cascade and RasGAP SH3 domain pathway.
- These pathways converge to regulate Mos protein synthesis, essential for Cdc2 activation and oocyte maturation.