The mammalian target of rapamycin (mTOR) governs cell growth and proliferation by mediating the mitogen- and nutrient-dependent signal transduction that regulates messenger RNA translation. We identified phosphatidic acid (PA) as a critical component of mTOR signaling. In our study, mitogenic stimulation of mammalian cells led to a phospholipase D-dependent accumulation of cellular PA, which was required for activation of mTOR downstream effectors. PA directly interacted with the domain in mTOR that is targeted by rapamycin, and this interaction was positively correlated with mTOR's ability to activate downstream effectors. The involvement of PA in mTOR signaling reveals an important function of this lipid in signal transduction and protein synthesis, as well as a direct link between mTOR and mitogens. Furthermore, these studies suggest a potential mechanism for the in vivo actions of the immunosuppressant rapamycin.

Summary The evolutionarily conserved target of rapamycin (TOR) signaling controls growth, metabolism and aging. In the first robust demonstration of pharmacologically-induced life extension in a mammal, longevity was extended in mice treated with rapamycin, an inhibitor of mechanistic TOR (mTOR). However, detrimental metabolic effects of rapamycin treatment were also reported, presenting a paradox of improved survival despite metabolic impairment. How rapamycin extended lifespan in mice with such paradoxical effects was unclear. Here we show that detrimental effects of rapamycin treatment were only observed during the early stages of treatment. As the treatment continued for 20 weeks, these effects were reversed or diminished; the mice had better metabolic profiles, increased oxygen consumption and ketogenesis, and markedly enhanced insulin sensitivity. Thus, prolonged rapamycin treatment led to beneficial metabolic alterations, consistent with life extension previously observed. Our findings provide a likely explanation of the “rapamycin paradox” and support the potential causal importance of these metabolic alterations in longevity.

Dishevelled (Dsh) is a cytoplasmic multidomain protein that is required for all known branches of the Wnt signalling pathway1–3. The Frizzled/planar cell polarity (Fz/PCP) signalling branch requires an asymmetric cortical localization of Dsh, but this process remains poorly understood. Using a genome-wide RNA interference (RNAi) screen in Drosophila melanogaster cells, we show that Dsh membrane localization is dependent on the Na+/H+ exchange activity of the plasma membrane exchanger Nhe2. Manipulating Nhe2 expression levels in the eye causes PCP defects, and Nhe2 interacts genetically with Fz. Our data show that the binding and surface recruitment of Dsh by Fz is pH- and charge-dependent. We identify a polybasic stretch within the Dsh DEP domain that binds to negatively charged phospholipids and appears to be mechanistically important. Dsh recruitment by Fz can be abolished by converting these basic amino-acid residues into acidic ones, as in the mutant, DshKR/E. In vivo, the DshKR/E(2×) mutant with two substituted residues fails to associate with the membrane during active PCP signalling but rescues canonical Wnt signalling defects in a dsh-background. These results suggest that direct interaction between Fz and Dsh is stabilized by a pH and charge-dependent interaction of the DEP domain with phospholipids. This stabilization is particularly important for the PCP signalling branch and, thus, promotes specific pathway selection in Wnt signalling.

The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact or integrate in the rapamycin-sensitive signaling network has not been examined before. Here, we find that PLD1, but not PLD2, is required for

Our observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA.