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A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity
M S Marshall1, W S Hill, A S Ng
1Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Abstract:
The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.
Insights
Researchers identified the C-terminal 343 amino acids of ras p21 GTPase activating protein (GAP) as the catalytic domain responsible for stimulating ras p21 GTPase activity. This region is crucial for GAP
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Ras GTPase activating protein (GAP) regulates ras p21 activity.
- Understanding GAP's functional domains is key to comprehending ras signaling pathways.
Purpose of the Study:
- To delineate the specific regions of bovine ras p21 GTPase activating protein (GAP) responsible for catalytic activity.
- To investigate the structural and functional domains of GAP.
Main Methods:
- Cloning and expression of bovine ras p21 GAP cDNA in Escherichia coli.
- Construction and expression of truncated GAP protein variants.
- Biochemical assays to measure ras p21 GTPase activity stimulation.
Main Results:
- The C-terminal 343 amino acids (residues 702-1044) of GAP possess full catalytic activity, binding Ha ras p21-GTP and stimulating GTPase activity.
- Deletion of the C-terminal 61 amino acids resulted in insoluble protein, indicating their importance for proper folding or stability.
- A homologous region in the N-terminal half of GAP, outside the catalytic domain, shows similarity to conserved domains in other signaling proteins.
Conclusions:
- A distinct catalytic domain resides at the C terminus of ras p21 GAP.
- GAP possesses at least two distinct domains: a C-terminal catalytic domain and an N-terminal domain with potential regulatory or interaction functions.