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Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain
E T Barfod1, Y Zheng, W J Kuang
1Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Abstract:
CDC42, a member of the Rho family of small GTP-binding proteins, regulates cytoskeletal rearrangements required for cell division. Activating mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-binding proteins, CDC42 is activated by a guanosine exchange factor and inactivated by a GTPase-activating protein (GAP). An unidentified 25-kDa platelet protein has been shown to function as a specific CDC42GAP. Here we report the cloning of a cDNA encoding this GAP from a human platelet-precursor cell line. Sequence analysis reveals the presence of three consensus box regions characteristic of rhoGAPs. A glutathione S-transferase fusion protein containing the three boxes derived from the new clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA clone encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85 alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.
Insights
Researchers identified a specific GTPase-activating protein (GAP) for CDC42, a protein crucial for cell division. This CDC42GAP may link CDC42 to other cellular signaling pathways, offering new insights into cell regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- CDC42, a Rho family GTPase, regulates cytoskeletal dynamics essential for cell division.
- Dysregulation of CDC42 activity, particularly through activating mutations, leads to abnormal cell phenotypes like multinucleation.
- GTPase-activating proteins (GAPs) inactivate small GTPases like CDC42, but the specific GAP for CDC42 in platelets was previously unidentified.
Purpose of the Study:
- To clone and characterize the cDNA encoding the specific 25-kDa CDC42 GTPase-activating protein (GAP) found in human platelets.
- To confirm the specificity of the cloned GAP for CDC42.
- To investigate potential interactions of the CDC42GAP with other signaling molecules.
Main Methods:
- Cloning of a cDNA encoding the CDC42GAP from a human platelet-precursor cell line.
- Sequence analysis to identify conserved domains characteristic of rhoGAPs.
- Expression of a glutathione S-transferase (GST) fusion protein containing key domains for functional assays.
- In vitro assays to measure the GTPase-activating protein activity of the fusion protein on CDC42 and other Rho proteins.
- Biochemical binding experiments to assess interactions with Src homology 3 (SH3) domains.
Main Results:
- Successful cloning of a cDNA encoding a specific CDC42GAP.
- Sequence analysis revealed three consensus box regions typical of rhoGAPs.
- The GST-fusion protein containing these boxes significantly stimulated CDC42 GTPase activity, demonstrating specificity.
- A proline-rich SH3-binding domain was identified, capable of interacting with SH3 domains of p85 alpha and c-Src.
Conclusions:
- The cloned cDNA encodes a specific GTPase-activating protein for CDC42.
- The identified CDC42GAP possesses an SH3-binding domain, suggesting a role in integrating CDC42 signaling with other pathways.
- This discovery provides a molecular link between CDC42 regulation and Src-family kinases and PI3K signaling.