Received for publication September 16, 2004.
Revised December 29, 2004.
Accepted for publication December 30, 2004.

Involvement of G_i/o proteins in nerve growth factor-stimulated phosphorylation and degradation of tuberin in PC12 cells and cortical neurons

Abstract

Tuberin is a critical translation regulator whose role in nerve growth factor (NGF)-promoted neuronal survival has not been documented. In the present study we examined the ability of NGF to regulate tuberin in PC12 cells and primary cortical neurons. Incubation of serum-deprived cells with NGF stimulated tuberin phosphorylation and induced proteosome-mediated tuberin degradation. Inhibition of the phosphatidylinositol-3-kinase (PI3K) by wortmannin or overexpression of the kinase dead Akt mutant completely blocked the NGF-induced tuberin phosphorylation and degradation. Interestingly, the NGF-induced tuberin phosphorylation was partially blocked by pertussis toxin or overexpression of regulators of G protein signaling (RGSZ1 and GAIP), suggesting the participation of G_i/o proteins. The use of transducin as a G scavenger indicated that G subunits rather than G_i/o acted as the signal transducer. Epidermal growth factor can similarly induce tuberin phosphorylation and degradation via a PI3K/Akt pathway in PC12 cells, but these responses were insensitive to pertussis toxin treatment. Treatment of PC12 cells with a specific agonist to the G_i-coupled ₂-adrenoceptor also stimulated tuberin phosphorylation transiently, further demonstrating the involvement of G_i/o signaling in tuberin regulation in PC12 cells. Finally, overexpression of non-phosphorylable tuberin attenuated NGF-promoted survival of PC12 cells, suggesting that the phosphorylation and degradation of tuberin are important for NGF-promoted cell survival. Collectively, this study demonstrates the regulatory effect of NGF and G_i/o signaling on tuberin.

Key words: NGF/EGF, Gi family, Protein Kinases (other), G protein regulation, RGS proteins, MAP Kinase