Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.

For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity, but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent labeled samples. While the popular LTQ linear ion trap supports analyzing iTRAQ reagent labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent labeled peptides, and increasing microscan acquisition and repeat count improves quantification, but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent labeled peptides, and provide guidelines for successful quantitative proteomic studies.

Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within this fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. This approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three dimensional peptide fractionation involving sequential steps of preparative IEF, strong cation exchange, and capillary reversed phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Cell growth relies on coordinated regulation of signaling pathways that integrate cellular physiological status in response to nutrient levels, growth factor signals, and environmental stress. Impairment of the coordinated regulation can lead to disastrous effects on cell physiology, resulting in cell death or uncontrolled growth. mTOR, 2 a member of the phosphatidylinositol kinase-related kinase family, has been known as a central player in the signaling pathway that regulates cell growth in response to a variety of cellular signals derived from nutrient levels, growth factors, and environmental stress (2-4). mTOR plays a central role in the signaling network that regulates a variety of cellular processes including ribosome biogenesis, protein synthesis, autophagy, and actin cytoskeleton organization; human diseases such as cancer, diabetes, obesity, and harmatoma syndrome are associated with defects in mTOR signaling (5-9).Recent years have seen discoveries of several mTOR effectors and binding proteins. mTOR exists in two multiprotein complexes, mTORC1 and mTORC2. mTORC1 consists of mTOR, raptor, G␤L, and PRAS40, and it functions to regulate protein synthesis and cell growth in response to nutrient levels and growth factor signals (10 -14). mTORC1 regulates phosphorylations of at least two regulators of protein synthesis, S6K1 and 4E-BP1, and mediates nutrient and insulin signals to the cell growth machinery (2, 15). mTORC1 is regulated by TSC-Rheb (tuberous sclerosis complex-Ras homolog-enriched in brain) signaling (16 -19). mTORC2 consists of mTOR, rictor, G␤L, and Sin1, and it does not likely bind rapamycin-FK506-binding protein 12 complex, which makes mTORC2 distinctive from mTORC1 (13,20,21). Saccharomyces cerevisiae TORC2 consists of TOR2, LST8, AVO1 (Sin1 ortholog), and AVO3 (rictor ortholog) and two other components, AVO2 and BIT61, whose homologues have not been identified in higher eukaryotes (13,22,23). Functions and regulatory mechanisms of mTORC2 remain largely unknown. Recent studies showed that mTORC2 regulates protein kinase C ␣ phosphorylation, actin cytoskeleton organization, and Akt phosphorylation at 21,24,25). Recognizing the complex relationship between mTOR, S6K1, and * This study was supported by the Tuberous Sclerosis Alliance, the Minnesota Medical Foundation, American Heart Association Grant 0655706Z, and National Institutes of Health Grant DK072004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in acc...

The modification of proteins by the cytotoxic, reactive aldehyde 4-hydroxynonenal (HNE) is known to alter protein function and impair cellular mechanisms. In order to identify susceptible amino acid sites of HNE modification within complex biological mixtures by microcapillary liquid chromatography and linear ion trap tandem mass spectrometry, we have developed a solid-phase capture and release strategy that utilizes reversible hydrazide chemistry to enrich HNE-modified peptides. To maximize the detection of fragment ions diagnostic of HNE modification, both neutral loss-dependent acquisition of MS/MS/MS spectra and the pulsed Q dissociation operation mode were employed. When the solid-phase hydrazide enrichment strategy was applied to a yeast lysate treated with HNE, 125 distinct amino acid sites of HNE modification were mapped on 67 different proteins. The endogenous susceptibility of many of these proteins to HNE modification was demonstrated by analyzing HNE-treated yeast cell cultures with a complementary biotin hydrazide enrichment strategy. Further analysis revealed that the majority of amino acid sites susceptible to HNE modification were histidine residues, with most of these sites being flanked by basic amino acid residues, and predicted to be solvent exposed. These results demonstrate the effectiveness of this novel strategy as a general platform for proteome-scale identification of amino acid sites susceptible to HNE modification from within complex mixtures.