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Ras GTPase-activating protein-associated p62 is a major v-Src-SH3-binding protein
1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Oncogenic transformation by v-Src is accompanied by marked morphological changes and cytoskeletal reorganization. Yet, the cytoskeleton-associated proteins with which v-Src interacts are largely unknown. We have studied the binding of v-Src-SH3 domain to cellular proteins utilizing a blot overlay procedure with a GST-v-Src-SH3 fusion protein as probe. A major 62-64 kDa v-Src-SH3-binding protein, present in detergent-insoluble cellular fractions, was identified as p21ras-GTPase-activating protein-associated p62 (GAPA62). In non-transformed cells, including NIH 3T3 cells, GAPA62 was present in both the RIP A-soluble and RIP A-insoluble fractions, but only the latter form was tyrosine-phosphorylated. In contrast, in polyoma middle T antigen-transformed 3T3 cells, GAPA62 was present only in the RIP A-insoluble fraction, where it was highly phosphorylated. It is suggested that tyrosine phosphorylation of GAPA62 may be an important determinant of its cellular localization and its possible function as a mediator of v-Src actions.
Insights
v-Src oncogenic transformation involves cytoskeletal changes. Researchers identified p21ras-GTPase-activating protein-associated p62 (GAPA62) as a key v-Src-SH3 binding protein, whose tyrosine phosphorylation affects its localization and function.
Area of Science:
- Cell Biology
- Molecular Oncology
- Protein Interactions
Background:
- Oncogenic transformation by v-Src causes significant cellular changes, including cytoskeletal reorganization.
- The specific cytoskeleton-associated proteins interacting with v-Src remain largely uncharacterized.
Purpose of the Study:
- To identify cellular proteins that bind to the v-Src-SH3 domain.
- To investigate the role of p21ras-GTPase-activating protein-associated p62 (GAPA62) in v-Src-mediated cellular changes.
Main Methods:
- Utilized a blot overlay assay with a GST-v-Src-SH3 fusion protein to probe for binding proteins.
- Analyzed the cellular localization and tyrosine phosphorylation status of GAPA62 in normal and transformed cells.
Main Results:
- Identified a 62-64 kDa protein, GAPA62, as a major v-Src-SH3 binding protein found in detergent-insoluble fractions.
- In non-transformed cells, GAPA62 was found in both soluble and insoluble fractions, with tyrosine phosphorylation occurring in the insoluble fraction.
- In polyoma middle T antigen-transformed cells, GAPA62 was exclusively in the insoluble fraction and highly tyrosine-phosphorylated.
Conclusions:
- Tyrosine phosphorylation of GAPA62 influences its cellular localization.
- GAPA62 is a potential mediator of v-Src actions, with its phosphorylation state critical for its function.