Target Of Rapamycin Complex 1 Activity Research Articles

Regulation of TORC1 by Rag GTPases in nutrient response

TORC1 (Target of rapamycin complex 1) has a critical role in the regulation of cell growth and cell size. A wide range of signals, including amino acids, is known to activate TORC1. Here, we report the identification of Rag GTPases as novel activators of TORC1 in response to amino acid signals. Knockdown of Rag gene expression suppressed the stimulatory effect of amino acids on TORC1 in Drosophila S2 cells. Expression of constitutively active (GTP-bound) Rag in mammalian cells enhances TORC1 in the absence of amino acids while expression of dominant negative Rag blocks the stimulatory effects of amino acids on TORC1. Drosophila genetic studies also show that the Rag GTPases regulate cell growth, autophagy, and animal viability under starvation. Together, our studies establish a novel function of Rag GTPases in TORC1 activation in response to amino acid signals.

Regulation of Ceramide Biosynthesis by TOR Complex 2

Ceramides and sphingoid long-chain bases (LCBs) are precursors to more complex sphingolipids and play distinct signaling roles crucial for cell growth and survival. Conserved reactions within the sphingolipid biosynthetic pathway are responsible for the formation of these intermediates. Components of target of rapamycin complex 2 (TORC2) have been implicated in the biosynthesis of sphingolipids in S. cerevisiae; however, the precise step regulated by this complex remains unknown. Here we demonstrate that yeast cells deficient in TORC2 activity are impaired for de novo ceramide biosynthesis both in vivo and in vitro. We find that TORC2 regulates this step in part by activating the AGC kinase Ypk2 and that this step is antagonized by the Ca2+/calmodulin-dependent phosphatase calcineurin. Because Ypk2 is activated independently by LCBs, the direct precursors to ceramides, our data suggest a model wherein TORC2 signaling is coupled with LCB levels to control Ypk2 activity and, ultimately, regulate ceramide formation.

Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth

Phosphatidylinositol-3-kinase (PI3K)/AKT signaling is essential for growth and metabolism and is elevated in many cancers. Enzymatic activity of AKT has been shown to depend on phosphorylation of two conserved sites by PDK1 and TOR (target of rapamycin) complex 2 (TORC2) in a PI3K-dependent manner. Here we analyze the role of TORC2-mediated AKT phosphorylation in Drosophila. Mutants removing critical TORC2 components, rictor and sin1, strongly reduced AKT hydrophobic motif (HM) phosphorylation and AKT activity, but showed only minor growth impairment. A mutant form of AKT lacking the HM phosphorylation site displayed comparable activity. In contrast to the mild effects of removing HM site phosphorylation at normal levels of PI3K activity, loss of TORC2 activity strongly inhibited hyperplasia caused by elevated pathway activity, as in mutants of the tumor suppressor PTEN. Thus, TORC2 acts as a rheostat to broaden the range of AKT signaling at the high end of its range.

TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity.

Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (Rheb) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of Rheb in TORC1 activation remains to be determined. However, the function of Rheb in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRheb has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRheb on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that Rheb does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of Rheb, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and Rheb have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.