Related Experiment Videos
Mutations in the SH3 domain of the src oncogene which decrease association of phosphatidylinositol 3'-kinase activity
D S Wages1, J Keefer, T B Rall
1Department of Microbiology, University of Virginia, Charlottesville 22908.
Abstract:
To analyze the signaling pathways utilized in malignant transformation by pp60v-src, we have isolated and characterized src mutants which possess normal levels of protein tyrosine kinase activity but which cause only a partially transformed phenotype. Our hypothesis is that such mutants are partially defective for transformation because they are defective in their ability to activate specific components of the cellular signaling machinery while still activating others. In this communication, we report on the molecular and biochemical characterization of one such mutant, CU12 (D. D. Anderson, R. P. Beckmann, E. H. Harms, K. Nakamura, and M. J. Weber, J. Virol. 37:455-458, 1981). Cells infected with this mutant are capable of anchorage-independent growth, but rather than exhibiting the rounded and refractile morphology characteristic of wild-type-infected cells, they display an extremely elongated, fusiform morphology. The morphological properties of this mutant src could be accounted for entirely by a single mutation in the SH3 domain (lysine 106 to glutamate). Other mutations were constructed in this region by in vitro mutagenesis, both in a v-src and in an activated c-src background, and several of them also induced a fusiform morphology. All of the mutations inducing fusiform morphology also resulted in decreased association of pp60src with phosphatidylinositol 3'-kinase activity. In addition, association of pp60src with some tyrosine-phosphorylated proteins was altered. We propose that the SH3 domain participates (along with the SH2 domain) in the interaction of pp60src with cellular signaling proteins, and we speculate that the association with phosphatidylinositol 3'-kinase plays an important role in the regulation of cellular morphology.
Insights
Investigating pp60v-src transformation, researchers found SH3 domain mutations alter cell shape and reduce phosphatidylinositol 3-kinase association. This suggests the SH3 domain is crucial for regulating cell morphology via signaling pathways.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncogenesis
Background:
- pp60v-src is a viral tyrosine kinase implicated in malignant transformation.
- Understanding its signaling pathways is key to deciphering cancer development.
- Mutants with partial transformation phenotypes offer insights into specific pathway contributions.
Purpose of the Study:
- To characterize src mutants with normal kinase activity but altered transformation phenotypes.
- To investigate the role of specific domains, like SH3, in pp60v-src-mediated signaling and transformation.
- To identify signaling components affected by these mutants.
Main Methods:
- Isolation and characterization of src mutants (e.g., CU12).
- In vitro mutagenesis to create mutations in the SH3 domain of v-src and c-src.
- Analysis of cellular morphology, anchorage-independent growth, and protein-protein interactions (pp60src association with phosphatidylinositol 3'-kinase and tyrosine-phosphorylated proteins).
Main Results:
- A specific SH3 domain mutation (Lys106 to Glu) in pp60v-src resulted in a fusiform cell morphology instead of the typical rounded shape.
- Several other SH3 domain mutations also induced fusiform morphology.
- Mutations causing fusiform morphology decreased the association of pp60src with phosphatidylinositol 3'-kinase activity and altered interactions with other tyrosine-phosphorylated proteins.
Conclusions:
- The SH3 domain of pp60src plays a critical role in regulating cellular morphology, likely by mediating interactions with signaling proteins.
- The SH2 and SH3 domains cooperate in pp60src's interaction with cellular signaling partners.
- Association with phosphatidylinositol 3'-kinase appears to be a significant factor in controlling cell shape during src-mediated transformation.