Related Experiment Videos
Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap
P van der Geer1, M Henkemeyer, T Jacks
1Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Abstract:
The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling.
Insights
p120-GTPase-activating protein (GAP) specifically downregulates the Ras/MAP kinase pathway after growth factor stimulation. Its absence increases Ras-GTP levels and prolongs MAP kinase activation, but does not affect mitogenic signaling or transformation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Signal Transduction
Background:
- Ras proteins act as molecular switches in signaling pathways downstream of protein-tyrosine kinases.
- GTPase-activating proteins (GAPs), such as p120-Gap and neurofibromin, stimulate Ras GTP hydrolysis, turning off the pathway.
- p120-Gap contains SH2 domains that recognize phosphotyrosine motifs, suggesting a role in coupling external signals to Ras regulation.
Purpose of the Study:
- To investigate the in vivo role of p120-Gap in Ras regulation.
- To determine the specific functions of p120-Gap in growth factor-stimulated signaling pathways.
- To analyze the impact of p120-Gap deficiency on Ras-GTP levels, MAP kinase activation, and downstream cellular responses.
Main Methods:
- Utilized fibroblasts from mouse embryos with a null mutation in the p120-Gap gene (Gap-/-).
- Stimulated wild-type and Gap-/- cells with platelet-derived growth factor (PDGF).
- Assessed Ras-GTP levels, mitogen-activated protein (MAP) kinase activation, DNA synthesis, and tyrosine phosphorylation of p190-rhoGap.
Main Results:
- PDGF stimulation of Gap-/- cells resulted in significantly higher Ras-GTP levels and prolonged MAP kinase activation compared to wild-type cells.
- The absence of p120-Gap did not affect DNA synthesis induction by PDGF or morphological transformation induced by v-src and EJ-ras oncogenes.
- Normal tyrosine phosphorylation of p190-rhoGap, a protein interacting with p120-Gap SH2 domains, was dependent on the presence of p120-Gap.
Conclusions:
- p120-Gap plays a specific role in downregulating the Ras/MAP kinase pathway following growth factor stimulation.
- p120-Gap modulates the tyrosine phosphorylation of p190-rhoGap.
- p120-Gap is not essential for all aspects of mitogenic signaling, such as DNA synthesis and oncogene-induced transformation.