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Published on: March 16, 2017
Visualization of phosphotyrosine containing molecules within the detergent insoluble cell matrix of v-src transformed
Abstract:
The RSV oncogene v-src is known to transform host cells through the action of a single gene protein product (pp60src) endowed with tyrosine specific kinase activity. The nature and cellular localization of substrates of pp60src kinase are largely unknown. Detergent insoluble cell matrix was prepared by treating RSV-transformed mouse fibroblasts with the non-ionic detergent NP-40. These preparations, highly enriched in cytoskeletal proteins, when observed in the E M consisted mainly of a meshwork of filaments; fragments of plasma membrane and nuclear "ghosts" were also present. Antibodies against phosphotyrosine -previously shown to be reactive with protein phosphorylated at tyrosine residues- were prepared and affinity purified using a synthetic hapten (azobenzyl phosphonic acid, ABP). By means of the immunogold techniques applied to electron microscopy, phosphotyrosine containing molecules were found to be present in RSV transformed, but not in control fibroblasts. Gold particles were mostly bound to electron dense granular material associated with the filaments. These results are consistent with the idea that pp60src itself and some tyrosine phosphorylated proteins may be found among detergent-insoluble cell structures.
Insights
Researchers identified tyrosine-phosphorylated proteins in detergent-insoluble cell structures of RSV-transformed fibroblasts. This finding sheds light on the cellular localization of substrates for the pp60src kinase, crucial in cell transformation.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- The Rous Sarcoma Virus (RSV) oncogene v-src transforms cells via its protein product, pp60src.
- pp60src possesses tyrosine-specific kinase activity.
- The substrates and cellular locations of pp60src kinase activity remain largely uncharacterized.
Purpose of the Study:
- To investigate the nature and cellular localization of pp60src kinase substrates.
- To identify tyrosine-phosphorylated proteins within RSV-transformed cells.
Main Methods:
- Preparation of detergent-insoluble cell matrix from RSV-transformed mouse fibroblasts using NP-40.
- Electron microscopy (EM) of the cell matrix to observe structural components.
- Production and affinity purification of anti-phosphotyrosine antibodies.
- Immunogold labeling and EM to detect phosphotyrosine-containing molecules.
Main Results:
- Detergent-insoluble preparations were enriched in cytoskeletal proteins, forming a filament meshwork.
- Phosphotyrosine-containing molecules were detected in RSV-transformed fibroblasts but not in control cells.
- Gold particles localized to electron-dense granular material associated with filaments.
Conclusions:
- Tyrosine-phosphorylated proteins are present in the detergent-insoluble fraction of RSV-transformed cells.
- pp60src and its substrates are likely associated with detergent-insoluble cellular structures.
- These findings contribute to understanding the mechanisms of RSV-induced cell transformation.
