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Epidermal growth factor-induced DNA synthesis. Key role for Src phosphorylation of the docking protein Gab2
Mei Kong1, Catherine Mounier, Victor Dumas
1Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada.
Abstract:
We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035-36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade.
Insights
Src family kinases phosphorylate Gab2, activating PI3-kinase signaling and DNA synthesis, crucial for epidermal growth factor (EGF)-induced liver cell growth. This pathway is essential for hepatic mitogenesis.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor (EGF) stimulates liver cell proliferation (mitogenesis) via phosphatidylinositol 3-kinase (PI3-kinase).
- A cytosolic complex containing Gab2 is responsible for most EGF-induced PI3-kinase activity, highlighting Gab2's critical role.
Purpose of the Study:
- To elucidate the role of Gab2 phosphorylation by Src family kinases in EGF-induced mitogenesis.
- To investigate the downstream signaling events mediated by Gab2 phosphorylation.
Main Methods:
- Utilized PP1, a Src family kinase inhibitor, to assess its effects on Gab2 phosphorylation and downstream signaling.
- Examined Gab2 phosphorylation in Csk knockout cells with constitutively active Src family kinases.
- Investigated the interaction between Gab2 and Src kinase using mutation analysis of Gab2's proline-rich sequences.
- Assessed the impact of Gab2 mutants lacking SHP2 binding sites on signaling pathways.
Main Results:
- PP1 inhibited EGF-induced Gab2 tyrosine phosphorylation, PI3-kinase activation, Akt phosphorylation, and DNA synthesis.
- Gab2 phosphorylation was increased in Csk knockout cells, confirming Src family kinase involvement.
- Gab2 and Src kinases constitutively associate, with proline-rich Gab2 sequences mediating this interaction.
- Mutating Gab2's proline-rich sites blocked EGF-induced signaling and mitogenesis.
- Overexpression of a Gab2 mutant lacking SHP2 binding sites enhanced PI3-kinase activation but inhibited MAPK activation, indicating SHP2's role in down-regulating Src-induced responses.
Conclusions:
- Src family kinase activation, through Gab2 phosphorylation, is essential for EGF-induced hepatic mitogenesis.
- The PI3-kinase cascade, activated via Gab2 phosphorylation, plays a central role in this process.
- Gab2 acts as a critical scaffold, integrating Src kinase activity into the PI3-kinase pathway for liver cell proliferation.