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Cloning of bovine GAP and its interaction with oncogenic ras p21
U S Vogel1, R A Dixon, M D Schaber
1Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Abstract:
The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
Insights
Researchers identified a GTPase-activating protein (GAP) that binds to active ras p21 protein complexes. This discovery sheds light on the regulation of normal and oncogenic ras proteins in cellular pathways.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Biochemistry
Background:
- Mammalian ras p21 proteins are key regulators in cellular signaling, sharing properties with G-proteins.
- Oncogenic ras p21 variants are constitutively active due to GTP binding.
- A cytosolic GTPase-activating protein (GAP) has been identified that modulates ras p21 activity.
Purpose of the Study:
- To characterize the interaction between bovine brain GAP and ras p21.
- To determine if GAP differentiates between normal and oncogenic ras p21.
- To clone and sequence the complementary DNA for bovine GAP.
Main Methods:
- Purification of bovine brain GAP.
- Kinetic competition assays using purified GAP and ras p21-GTP/GDP complexes.
- Complementary DNA cloning and sequencing of bovine GAP.
Main Results:
- Bovine brain GAP (125,000 Da) was identified and purified.
- GAP preferentially interacts with the active GTP-bound forms of both normal and oncogenic Harvey (Ha) ras p21.
- The complementary DNA for bovine GAP was cloned and sequenced, revealing conserved domains with other signaling proteins.
Conclusions:
- Bovine GAP interacts with active ras p21, suggesting a role in regulating ras signaling.
- GAP's interaction with both normal and oncogenic ras p21 provides insights into its regulatory mechanism.
- The sequence homology of GAP suggests evolutionary conservation and potential involvement in diverse signaling pathways.