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Original Article

Involvement of G_i/o Proteins in Nerve Growth Factor-Stimulated Phosphorylation and Degradation of Tuberin in PC-12 Cells and Cortical Neurons

Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

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 PC-12 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. It is interesting that the NGF-induced tuberin phosphorylation was partially blocked by pertussis toxin or overexpression of regulators of G protein signaling (regulator of G protein signaling Z1 and G-interacting protein), 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 PC-12 cells, but these responses were insensitive to pertussis toxin treatment. Treatment of PC-12 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 PC-12 cells. Finally, overexpression of nonphosphorylable tuberin attenuated NGF-promoted survival of PC-12 cells, suggesting that the phosphorylation and degradation of tuberin are important for NGF-promoted cell survival. Together, this study demonstrates the regulatory effect of NGF and G_i/o signaling on tuberin.

Address correspondence to: Dr. Yung H. Wong, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: boyung{at}ust.hk