Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, senses and integrates signals from diverse environmental cues (14, 31, 50, 74). mTOR associates with different partner proteins to form functionally distinct signaling complexes (4). The immunosuppressive drug rapamycin acutely inhibits signaling by mTOR complex 1 (mTORC1) (22), which contains mTOR, mLST8/GβL, raptor, and PRAS40 (24, 33, 34, 54, 67). Rapamycin fails to acutely inhibit signaling by mTORC2, which contains mTOR, mLST8/GβL, rictor, mSin1, and PRR5/Protor (18, 32, 47, 55, 73, 76). mTORC1 promotes various biosynthetic processes, including protein synthesis, cell growth (an increase in cell mass and size), and cell proliferation (an increase in cell number) (14, 40, 74). During growth factor (e.g., insulin) and nutrient (e.g., amino acids and glucose) sufficiency, mTORC1 phosphorylates the translational regulators p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) to coordinately upregulate protein biosynthesis (40). Both S6K1 and 4EBP1 contain a TOR signaling motif, which mediates their interaction with raptor and thus facilitates their recruitment to the mTOR kinase (10, 44, 57, 58). In addition to regulating protein synthesis, mTORC1-mediated phosphorylation of S6K1 and 4EBP also promotes cell growth and cell cycle progression (15, 16). While more recently identified and thus less well characterized than mTORC1, mTORC2 mediates the phosphorylation of AGC kinase family members (e.g., Akt [also known as protein kinase B, PKB], PKCα, and SGK1) on their hydrophobic motifs and modulates the organization of the actin cytoskeleton (20, 26, 32, 55, 56).The insulin pathway represents the best-characterized activator of mTORC1 signaling to date, and thus many signaling intermediates that link insulin receptor activation to mTORC1 have been identified (12, 31). Complementary work using Drosophila melanogaster genetics and mammalian cell culture identified TSC1 (hamartin) and TSC2 (tuberin) as upstream negative regulators of mTORC1 (27). Inactivation of either the TSC1 or TSC2 genes, whose protein products heterodimerize to form a tumor suppressor complex, causes the development of benign tumors in diverse organs in both humans and rodents, a disease known as tuberous sclerosis complex (TSC) (36). TSC2 contains a GTPase-activating protein domain that acts on Rheb, a Ras-like GTP binding protein that activates mTORC1 (27). Thus, in TSC-deficient cells, constitutive Rheb-GTP leads to chronically high mTORC1 signaling. While the mechanism by which Rheb-GTP activates mTORC1 remains incompletely understood, Rheb coimmunoprecipitates with mTOR and directly activates mTORC1 kinase activity in vivo and in vitro when GTP bound (2, 38, 54). Rheb has been reported to augment the activity of PLD1, an enzyme that catalyzes the production of the lipid second messenger phosphatidic acid, which contributes to the mitogenic activation of mTORC1 signaling (13, 62). Additionally, Rheb-GTP was reported to induce the dissociation of the endogenous mTOR inhibitor FKBP38 (3), although aspects of this model have been questioned (72). Insulin/phosphatidylinositol 3-kinase (PI3K) signaling reduces the inhibitory effect of TSC on mTORC1 via Akt-mediated phosphorylation of TSC2 (29, 42, 64). Additionally, Ras-regulated signaling via mitogen-activated protein kinase (MAPK) and RSK also inhibits TSC via PI3K/Akt-independent phosphorylation of TSC2 (39, 51, 63). In contrast, glucose deprivation enhances TSC''s inhibitory effect on mTORC1 signaling via AMP-activated protein kinase (AMPK)-mediated phosphorylation of TSC2 (on different sites) (30). Thus, TSC functions as a central nexus of diverse physiological signals to fine-tune mTORC1 signaling depending on environmental conditions (27). While the mechanism by which amino acids promote mTORC1 signaling has remained elusive, compelling new data reveal that the Rag GTPases link amino acid sensing to mTORC1 activation (35, 52, 53). During amino acid sufficiency, GTP-bound Rag heterodimers bind raptor and recruit mTORC1 to an endomembrane compartment that contains the mTORC1 activator Rheb; thus, amino acid sufficiency may function to prime mTORC1 for subsequent growth factor-mediated activation via a dynamic subcellular redistribution mechanism (52).Despite the well-characterized regulation of mTORC1 signaling by growth factors (e.g., insulin), nutrients (e.g., amino acids and glucose), and cellular stress (e.g., hypoxia) and the identification of numerous signaling mediators of these pathways, the direct molecular mechanisms by which cellular signals modulate mTORC1 action remain obscure (31). While three phosphorylation sites (P-sites) on mTOR have been reported to date (T2446, S2448, and S2481), no function has yet been ascribed to any site (7, 43, 49, 59). Here we identify S1261 as a novel mTOR phosphorylation site in vivo in cultured mammalian cells and provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function. We show that insulin signals via the PI3K/TSC/Rheb pathway in an amino acid-dependent and rapamycin-insensitive manner to promote mTOR S1261 phosphorylation, which regulates mTORC1 autokinase activity, biochemical signaling to downstream substrates, and cell growth to increased cell size, a major cellular function of mTORC1. Elucidation of the molecular mechanisms underlying mTORC1 regulation will enable us to better understand how mTORC1 senses environmental stimuli to control cellular physiology. As aberrantly upregulated mTORC1 signaling likely contributes to cancer, insulin-resistant diabetes, and cardiovascular diseases, understanding mTORC1 regulation may aid in the development of novel therapeutics for these prevalent human diseases (11, 21, 28).     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

Asymmetric DNA requirements in Xer recombination activation by FtsK

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis.     

Surveillance of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in dairy goat herds

This study was designed to monitor the presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) in 66 dairy goat herds of a genetic improvement programme in a region of Spain where contagious agalactia is endemic. Over a whole lactation period, 300 bulk tank milk and 381 milk samples from goats with clinical mastitis were subjected to polymerase chain reaction (PCR) to detect the two mycoplasma species. The presence of mycoplasmas (either species or both) was detected in 66.7% of the herds and M. agalactiae was identified in 95.45% of these positives herds. In a given infected herd, mycoplasmas were not continuously detected over the whole study period. Our findings indicate that in an endemic area, M. agalactiae and Mmc can be monitored through PCR analysis of mastitic milk and bulk tank milk (BTM) samples. Over a lactation period we recommend testing multiple BTM samples on a herd. No relationship was observed between the use of inactivated mycoplasma vaccines and the PCR detection of both mycoplasmas.     

Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus

The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5' untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5' untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the lambda integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.     

Inactivated vaccine induces protection against Mycoplasma agalactiae infection in sheep

The efficacy of an inactivated oil-emulsion vaccine against Mycoplasma agalactiae was evaluated by an experimental infection of sheep. The vaccinated sheep developed high levels of antibodies and, following challenge, they did not develop any clinical signs of disease and the mycoplasmas were not detected, either by isolation trials or PCR assays carried out both on nasal swabs and milk specimens. The unvaccinated-challenged sheep showed typical signs of M. agalactiae infection and bacterial shedding. The results obtained indicate a good efficacy of the vaccine in eliciting protection against M. agalactiae infection.     

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.     

Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization

Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.     

An oil-emulsion vaccine induces full-protection against Mycoplasma agalactiae infection in sheep

The immunogenicity and efficacy of three inactivated vaccines (A, B, C) prepared with Mycoplasma agalactiae (M. agalactiae) and with different oil-emulsion adjuvants were evaluated in sheep. Twenty-eight animals were used, divided into four groups (a, b, c, d) of seven animals each. Three groups were immunized with the same vaccine, but using different adjuvants, while one group (d) was used as an unvaccinated control group. All the vaccine formulations were able to induce clinical protection of animals after challenge with M. agalactiae, but only vaccine C, emulsioned with Montanide ISA-563, Marcol-52 and Montane-80 (ratio: 30%, 63%, 7% respectively), was able to induce full protection in challenged animals, preventing both the onset of clinical signs and infection by M. agalactiae.     

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens,Mycoplasma bovis and Mycoplasma agalactiae

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.     

DNA Inversions between Short Inverted Repeats in Escherichia Coli

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.     

Site-Specific Formation of A basic Lesions in DNA

Abstract

A method for the introduction of depurinated lesions in DNA is described, and is based on the formation of a covalent cross-link between an antisense oligonucleotide probe and the target DNA sequence followed by an unexpectedly mild thermal depurination.     

Inversions in mitochondrial DNA of a petite mutant of yeast

Mitochondrial DNA of an erythromycin-resistant petite mutant of yeast, E734, showed physical maps of inversion, which occurred between two cross-over sites in the fragments Hae A and Hae B. The pair of cross-over sites was inferred to be accommodated within a repeat unit of E734 mtDNA during petite mutation by joining two fragments excised from non-adjacent region of wild type mtDNA.     

DNA methylation and Mycoplasma genomes

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.     

A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms

An integrative conjugative element, ICEA, was characterized in Mycoplasma agalactiae strain 5632, in which it occurs as multiple chromosomal copies and as a free circular form. The distribution of ICEA sequences in M. agalactiae strains and their occurrence in Mycoplasma bovis suggest the spreading of the element within or between species.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.