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A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42
E Manser1, T Leung, H Salihuddin
1Institute of Molecular and Cell Biology, National University of Singapore.
Abstract:
The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs that recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.
Insights
Researchers identified a hippocampal tyrosine kinase that binds to the active, GTP-bound form of Cdc42Hs. This interaction inhibits GTP hydrolysis, suggesting a regulatory mechanism sustaining Cdc42Hs activity via tyrosine phosphorylation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Ras-related Rho subfamily of GTP-binding proteins (p21s), including Rho, Rac, and Cdc42Hs, are crucial for cytoskeletal organization.
- These proteins function similarly to p21ras, with their active GTP-bound state inactivated by GTP hydrolysis, a process often stimulated by GTPase-activating proteins (GAPs).
- Previous research identified diverse GAPs for this subfamily, such as n-chimaerin in the hippocampus, and proteins that inhibit GTP hydrolysis.
Purpose of the Study:
- To characterize a novel hippocampal protein that interacts with Cdc42Hs.
- To elucidate the mechanism by which this interaction affects Cdc42Hs activity.
- To explore potential regulatory pathways involving tyrosine phosphorylation and Cdc42Hs.
Main Methods:
- Characterization of a hippocampal complementary DNA (cDNA) encoding a tyrosine kinase.
- Analysis of the binding specificity of the tyrosine kinase to different forms of Cdc42Hs.
- Assays to measure the effect of the tyrosine kinase on intrinsic and GAP-stimulated GTPase activity of Cdc42Hs.
Main Results:
- A hippocampal tyrosine kinase was identified that specifically binds to the GTP-bound (active) form of Cdc42Hs.
- This binding is mediated by a unique 47-amino acid sequence C-terminal to an SH3 domain.
- The tyrosine kinase binding inhibits both the intrinsic and GTPase-activating protein (GAP)-stimulated GTP hydrolysis of Cdc42Hs.
Conclusions:
- A novel regulatory mechanism may exist to sustain the active, GTP-bound form of Cdc42Hs in the hippocampus.
- This mechanism is directly linked to a tyrosine phosphorylation pathway.
- The identified tyrosine kinase plays a role in regulating Cdc42Hs activity, impacting cytoskeletal organization pathways.