mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin-with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1␣ and HIF2␣ has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2␣ expression is the more important contributor. Expression of HIF1␣ is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2␣ expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1␣ and HIF2␣ to rapamycin, both HIF1␣ and HIF2␣ expression is dependent on mTOR. HIF1␣ expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2␣ was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1␣ is dependent on both mTORC1 and mTORC2, HIF2␣ is dependent only on mTORC2. We also examined the dependence of HIF␣ expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2␣ was dependent on Akt2, whereas that of HIF1␣ was dependent on Akt3. Because HIF2␣ is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2␣ has been implicated. Hypoxia-inducible factor (HIF)2 is a critical transcriptional regulator of cellular responses to a variety of stressful conditions (1, 2). Under non-stressful conditions, HIF␣ is ubiquitinated by the von Hippel-Lindau (VHL) gene product pVHL, a substrate-conferring component of a ubiquitin-protein isopeptide ligase that targets HIF␣ for degradation by the proteasome (3). Loss of the VHL gene results in a variety of pathologies, most significantly renal cell carcinoma (RCC) (4 -6). In the absence of pVHL, there is an up-regulation of HIF␣, and elevated expression of HIF␣ has been strongly implicated in VHL disease and RCC (4 -6). HIF␣ dimerizes with HIF␤ to form a transcription factor HIF that stimulates the transcription of genes that regulate angiogenesis and other factors important for responding to hypoxic and other stressful conditions such as vascular endothelial growth factor and glycolytic enzymes (2,7,8). There are several distinct ␣-subunits, but it is the expression of HIF1␣ and HIF2␣ that is most frequently elevated in human cancers (4, 9). Whereas HIF1␣ has both pro-and anti-proliferative properties, HIF2␣ lacks the anti-proliferative properties and is more strongly implicated in tumorigenesis (10). The somewhat overlapping and antagonistic effects of HIF1␣ and HIF2␣ are poorly understood, but it is clear that in RCC, HIF2␣ is a critical factor in that suppression of HIF2␣ blocks tumor formation by renal cancer cells (11,12). It is believed that the elevated expression of HIF2␣ contributes to the survival signals in rena...

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F-box protein βTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the βTrCP degron in DEPTOR is executed by CK1α, after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the βTrCP-dependent degradation of DEPTOR via βTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind βTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and βTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR signaling pathways.