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Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src
M Grossi1, S A La Rocca, G Pierluigi
1Istituto Pasteur-Fondazione Cenci-Bolognetti, Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma La Sapienza, Italy.
Abstract:
Quiescent mammalian fibroblasts can be induced to reenter the cell cycle by growth factors and oncoproteins. We studied the pathway(s) through which v-Src, the oncogenic tyrosine kinase encoded by the v-src oncogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to enter S-phase. To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele from the mutant ts LA31 PR-A. NIH(MR31) cells displayed a temperature-conditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature. Serum stimulation or thermolabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kinases with very little tyrosine phosphorylation of the Shc adaptor proteins at least during the early stages of v-Src reactivation and that v-Src-induced S-phase entry was strongly inhibited by drugs affecting MEK or PI 3-kinase. Our results also suggest that down-regulation of gas1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 down-regulation does not require PI 3-kinase dependent signals.
Insights
Oncoprotein v-Src drives quiescent cells into S-phase by activating Erk1/Erk2 MAP kinases and down-regulating Gas1. This process is inhibited by MEK or PI 3-kinase drugs, revealing key cell cycle entry pathways.
Area of Science:
- Cell Biology
- Molecular Oncology
- Signal Transduction
Background:
- Quiescent mammalian fibroblasts can reenter the cell cycle upon stimulation by growth factors or oncoproteins.
- The v-Src tyrosine kinase, encoded by Rous sarcoma virus, is a potent oncogene that can induce cellular transformation and proliferation.
Purpose of the Study:
- To investigate the molecular pathways by which the oncogenic tyrosine kinase v-Src induces quiescent NIH3T3 fibroblasts to enter S-phase.
- To characterize the role of specific signaling molecules, such as MAP kinases and PI 3-kinase, in v-Src-mediated cell cycle reentry.
Main Methods:
- Isolation and characterization of NIH3T3 cells expressing a temperature-sensitive v-src allele (NIH(MR31) cells).
- Induction of quiescence by serum deprivation at a restrictive temperature.
- Stimulation of cell cycle reentry by serum or v-Src reactivation.
- Pharmacological inhibition of MEK and PI 3-kinase pathways.
- Analysis of Erk1/Erk2 MAP kinase activation and Shc protein phosphorylation.
- Assessment of gas1 gene expression levels.
Main Results:
- Serum stimulation or v-Src reactivation induced S-phase entry with distinct kinetics.
- v-Src-induced mitogenesis involved early activation of Erk1/Erk2 MAP kinases.
- Early stages of v-Src reactivation showed minimal Shc protein tyrosine phosphorylation.
- Inhibition of MEK or PI 3-kinase significantly blocked v-Src-induced S-phase entry.
- Down-regulation of gas1 gene expression was crucial for efficient S-phase entry triggered by v-Src.
- Gas1 down-regulation did not necessitate PI 3-kinase-dependent signals.
Conclusions:
- v-Src utilizes the Erk1/Erk2 MAP kinase pathway for mitogenic activity, influencing cell cycle reentry.
- The PI 3-kinase pathway also plays a significant role in v-Src-induced S-phase entry.
- Down-regulation of the gas1 gene is a critical event in v-Src-mediated cell cycle progression, independent of PI 3-kinase signaling.