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pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide
1Department of Medicine, College of Medicine, Pennsylvania State University, Hershey 17033, USA.
Abstract:
Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.
Insights
Mutating the src homology 2 (SH2) domain of pp60v-src affects cell transformation. Low-affinity phosphopeptide binding correlates with rat cell transformation, suggesting substrates for pp60v-src in rat cells interact weakly.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- The substrates and precise role of the src homology 2 (SH2) domain in pp60v-src-mediated cell transformation are not fully understood.
- Investigating the SH2 domain's function is crucial for understanding viral oncogenesis and identifying therapeutic targets.
Purpose of the Study:
- To investigate the role of the SH2 domain in pp60v-src-mediated cell transformation by creating and analyzing mutant v-src alleles.
- To determine the correlation between phosphopeptide binding affinity and host cell transformation phenotypes.
Main Methods:
- Site-directed mutagenesis was employed to generate mutant v-src alleles with altered SH2 domain phosphotyrosine-binding capabilities.
- Mutant v-src proteins were analyzed for their ability to transform chicken and rat cells, and their phosphopeptide binding affinities were assessed in vitro.
Main Results:
- Mutations at Arginine-175 in the SH2 domain resulted in host-dependent transformation, with altered morphology in chicken cells (round) compared to known mutations (fusiform).
- In vitro phosphopeptide binding affinity correlated with specific cell transformation phenotypes: high affinity for chicken cell transformation and low affinity for rat cell transformation.
- The study identified a correlation between low-affinity phosphopeptide binding and rat cell transformation efficiency.
Conclusions:
- The SH2 domain's phosphopeptide binding affinity is critical for determining the host cell specificity of pp60v-src transformation.
- Identifying substrates that interact with pp60v-src with low affinity is a promising strategy for understanding rat cell transformation.