Class III phosphoinositide 3 -kinase is a nutrient sensor required for activation of p70 S6 kinase

Abstract

Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6Kl. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI(3)P, or siRNA knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as siRNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Regulation of hVps34 by starvation may be through phosphorylation. hVps34 is a phosphoprotein. Furthermore, hVps34 in vitro activity is negatively regulated by PP2A phosphatase. Finally, point mutagenesis of a prospective phosphorylation site in the C-terminal kinase domain of hVps34 regulates in vitro activity. Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.