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Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus
S Park1, M S Marshall, J B Gibbs
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109.
Abstract:
Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by oncogenic viral Src (v-Src) and that GAP is associated with a complex containing normal cellular Src (c-Src) in vertebrate fibroblasts. To investigate molecular interactions between the Src kinases and GAP, we developed an in vitro system for reconstituting Src-GAP complexes. For this purpose, we constructed recombinant baculovirus vectors that direct expression of Rous sarcoma virus v-Src, chicken c-Src, and bovine GAP in infected Sf9 insect cells. In vitro reconstitution experiments using baculovirus-expressed proteins demonstrate that both v-Src and c-Src associate in complexes with GAP. In addition, in vitro and in vivo phosphorylation analyses indicate that GAP serves as a substrate for both the v-Src and c-Src tyrosine kinases. To determine which structural features of GAP are involved in interactions with the Src kinases, we constructed recombinant baculoviruses that encode deletion mutants of bovine GAP. Deletion of the GAP amino-terminal portion containing Src homology 2 regions, which are highly conserved structural motifs postulated to mediate interactions among proteins, diminishes GAP phosphorylation and association with Src. This reconstitution system should facilitate further studies of molecular interactions between the Src kinases and GAP.
Insights
Ras GTPase-activating protein (GAP) interacts with and is phosphorylated by Src kinases. Deleting specific GAP regions reduces these interactions, aiding the study of Src-GAP molecular dynamics.
Area of Science:
- Molecular biology
- Cell signaling
- Biochemistry
Background:
- Ras GTPase-activating protein (GAP) is crucial for signal transduction pathways involving receptor tyrosine kinases and oncogenic Src.
- Previous research suggested interactions between Src kinases and GAP in vertebrate cells.
Purpose of the Study:
- To investigate the molecular interactions between Src kinases and GAP.
- To establish an in vitro system for reconstituting Src-GAP complexes.
Main Methods:
- Developed recombinant baculovirus vectors to express v-Src, c-Src, and bovine GAP in Sf9 insect cells.
- Utilized in vitro reconstitution experiments with baculovirus-expressed proteins.
- Performed in vitro and in vivo phosphorylation analyses and constructed GAP deletion mutants.
Main Results:
- Demonstrated that both v-Src and c-Src associate with GAP in reconstituted complexes.
- Confirmed GAP as a substrate for both v-Src and c-Src tyrosine kinases.
- Showed that deletion of the N-terminal region of GAP, including Src homology 2 domains, reduces GAP phosphorylation and Src association.
Conclusions:
- The study successfully established a system for reconstituting Src-GAP complexes, facilitating further research.
- Identified specific structural features of GAP, particularly the N-terminal region with SH2 domains, as critical for Src interaction and phosphorylation.