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Association of a tyrosine kinase activity with GAP complexes in v-src transformed fibroblasts
1Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Abstract:
p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.
Insights
v-src-transformed fibroblasts show p21ras GAP protein associated with tyrosine-phosphorylated proteins. A tyrosine kinase within GAP immunoprecipitates phosphorylates GAP, p62, and p190, potentially identifying v-src as the responsible kinase.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncogenesis
Background:
- p21ras GTPase-activating protein (GAP) is crucial in regulating ras signaling pathways.
- Tyrosine phosphorylation of GAP is implicated in cellular transformation, particularly in cells expressing viral sarcoma (v-src) kinase.
- GAP associates with other tyrosine-phosphorylated proteins in v-src-transformed cells, suggesting a complex regulatory network.
Purpose of the Study:
- To identify the specific tyrosine kinase responsible for phosphorylating p21ras GAP and its associated proteins in v-src-transformed fibroblasts.
- To investigate the in vivo relevance of the identified kinase activity.
Main Methods:
- Immunoprecipitation of GAP from v-src-transformed fibroblasts.
- In vitro kinase assays using immunoprecipitated proteins.
- Tryptic peptide analysis to compare in vitro and in vivo phosphorylation sites.
- In vitro association assays between GAP and pp60v-src.
Main Results:
- GAP immunoprecipitates from v-src-transformed cells exhibit tyrosine kinase activity.
- This activity phosphorylates GAP, as well as associated 62 kDa (p62) and 190 kDa (p190) proteins.
- Phosphorylation sites identified by tryptic peptide analysis are identical for in vitro and in vivo phosphorylation, suggesting the precipitated kinase is active in vivo.
- pp60v-src associates with GAP in vitro and can phosphorylate GAP in immune complex assays, indicating v-src is a likely candidate kinase.
Conclusions:
- A tyrosine kinase activity associated with p21ras GAP in v-src-transformed cells phosphorylates GAP, p62, and p190.
- The viral sarcoma (v-src) kinase (pp60v-src) is identified as a strong candidate responsible for the observed tyrosine phosphorylation of GAP in vivo.
- These findings elucidate a key mechanism in v-src-mediated cellular transformation involving direct regulation of GAP activity.