The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.Cell growth and proliferation are fundamental processes that are regulated by multiple signals, such as nutrients, hormones, and growth factors. One of the key components regulating these signal transduction pathways is mTOR, 3 which in higher eukaryotes forms two different complexes: mTORC1 and mTORC2 (1, 2). mTOR complexes display Ser/Thr kinase activity. mTORC1 is rapamycin-sensitive and includes the mTOR catalytic subunit, mLST8/G␤L, PRAS40, the regulatoryassociated protein of mTOR (raptor), and DEPTOR (3-5). Instead, mTORC2 is rapamycin-insensitive, at least in short treatments (6), and includes mTOR, mLST8/G␤L, mSin1, rapamycin-insensitive companion of mTOR (rictor), the protein observed with rictor (protor), and DEPTOR (4, 5, 7). mTORC1 regulates cell growth by controlling mRNA translation, ribosome biogenesis, autophagy, and metabolism (1, 8 -12). On the other hand, mTORC2 regulates cell survival and proliferation (13-18). Both hormones and growth factors have been described to regulate mTORC1 and mTORC2 (1,19,20). However, thus far only mTORC1 has been described to be regulated by nutrients such as amino acids (1, 2, 20 -22).A number of studies point to the importance of nutrients in the etiology of some major diseases, such as insulin-resistant obesity (23) or cancer (24, 25) and in the aging process (11,26). In humans, circulating amino acid levels are elevated in obese individuals, and this is related to an increase in insulin resistance (23,27,28). The morbidity of obesity not only extends to diabetes and cardiovascular diseases but also has been linked to 20% of cancer deaths (29). During the last years, different studies have shown a relationship between nutrients, such as glucose or amino acids, and mTOR signaling. For example, it has been reported that amino acids activate S6 kinase 1 protein (S6K1) and inhibit autophagy in an mTOR-dependent manner (30, 31). S6K1 activati...

Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwB⌬N70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwB⌬N70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwB⌬N70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwB⌬N70 -DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F 1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor. molecular motor ͉ DNA transfer C onjugative plasmids provide a main route for acquisition of new genetic information in bacteria. Bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show similarities to both DNA replication and protein transport systems (1, 2). The translocated substrate is a nucleoprotein particle that crosses the bacterial envelope into other bacterial or eukaryotic cells, crossing the kingdom boundaries. A two-step mechanism for DNA transport was proposed (2), in which conjugation is visualized as a DNA rolling-circle replication process linked to a type IV protein secretion system. Conjugation is encoded by two gene clusters in most conjugative plasmids. One cluster (dtr) encodes the DNA transfer replication proteins, and the other (mpf ) codes for the proteins that assemble the type IV protein secretion system channel. R388 is a 34-kb plasmid, which displays the simplest known dtr region, encoding only three proteins (TrwA, TrwB, and TrwC). The mpf region codes for 11 proteins (TrwD-N) involved in the formation of an extracellular pilus and a membrane-associated complex that translocates the nucleoprotein substrate across membranes.TrwB, an integral membrane and DNA-binding protein, is a key player in R388 conjugation (3-6). It has a counterpart in all conjugative systems, and it is thought to be responsible for recruiting the relaxosome DNA-pr...

The ribosomal protein S6 kinase 1 (S6K1) is emerging as a common downstream target of signalling by hormones and nutrients such as insulin and amino acids. Here, we have investigated how amino acids signal through the S6K1 pathway. First, we found that a commercial anti-phospho-Thr389-S6K1 antibody detects an 80-90 kDa protein that is rapidly phosphorylated in response to amino acids. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI-3 kinase inhibitors, and knockdown experiments showed that this protein was not S6K1. Looking for candidate targets of this phosphorylation, we found that amino acids stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. In turn, these phosphorylations required the activity of either p38 or ERK MAP kinases, which could compensate for each other. Moreover, we show that these MAP kinases are also needed for the amino acid-induced phosphorylation of S6K1 at Thr421/Ser424, as well as for that of S6K1 substrate, the S6 ribosomal protein. Consistent with these results, concomitant inhibition of p38 and ERK pathways also antagonised the well-known effects of amino acids on the process of autophagy. Altogether, these findings demonstrate a previously unknown role for MAP kinases in amino acid signalling.

Background: Metabolites may activate signal transduction pathways that regulate cell metabolism. Results: Amino acids activate Fru-2,6-P 2 synthesis through Akt-dependent phosphorylation of a specific PFKFB2 isoform. Conclusion: Amino acids regulate Fru-2,6-P 2 metabolism via Akt signaling. Significance: This study shows how signaling and metabolism are inextricably linked.