The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth. In the budding yeast Saccharomyces cerevisiae, TOR is found in two distinct multi-protein complexes 1,2 . TORC1 contains LST8, KOG1 and either TOR1 or TOR2 (that is, either one of the two TOR homologues in yeast). TORC1 couples transcription, ribosome biogenesis, translation initiation, nutrient uptake and autophagy to the abundance and quality of available nutrients. Thus it functions as a temporal regulator of cell growth 3 . The immunosuppressive and anti-cancer drug rapamycin binds directly to TOR1 or TOR2 in TORC1 and thereby inhibits TORC1 signalling. TORC2 contains LST8, AVO1, AVO2, AVO3 and TOR2. TORC2 mediates spatial control of cell growth by polarizing the actin cytoskeleton, and thus the secretory pathway, towards the bud or growth site. TORC2 signalling is rapamycin insensitive owing to the inability of rapamycin to bind to TOR2 in TORC2 (ref. 1). Like TOR, LST8 and KOG1 have obvious sequence homologues in all eukaryotic genomes examined, and mammalian TOR (mTOR) forms a rapamycin-sensitive, TORC1-like complex with mLST8 (GβL) and raptor (mKOG1) 1,4-6 . Sequence homologues of the AVOs are less obvious or have been absent in earlier mammalian databases, and thus the existence of an mTORC2 has yet to be demonstrated. Searches of more recent and complete versions of mammalian genomes revealed fragments of a probable vertebrate homologue of the TORC2-specific protein AVO3. Mammalian AVO3 (mAVO3) was cloned from mouse C2C12 cDNA and sequenced, revealing a putative protein of 1,708 amino acids that shares about 25% identity with S. cerevisiae AVO3. Although there are no obvious functional domains in mAVO3, there are six regions that seem to be more highly conserved in Saccharomyces cerevisiae, Dictyostelium discoideum, Drosophila melanogaster and Mus musculus We wished to determine if mAVO3 shares functional similarity with yeast AVO3. Searches using BLAT (the BLAST-like alignment tool) predicted an mAVO3 transcript of about 9.5 kilobases (kb). A roughly 9.5-kb transcript was detected in all tissues examined, with a higher signal observed in skeletal muscle, kidney, placenta and leukocytes (data not shown). This tissue expression profile closely resembles that observed for the mRNAs encoding mTOR, raptor and mLST8 (refs 1,5), suggesting that mAVO3 may interact with at least some of these proteins. To test if endogenous mAVO3 forms a complex with mTOR, raptor and/or mLST8, rabbit antisera were generated that specifically recognize mAVO3 and raptor. Immunoprecipitation of mTOR from HEK293 cells co-precipitated both raptor and mAVO3 We next wished to determine if mTORC2, like yeast TORC2, is insensitive to rapamycin. In yeast and mammalian cells, rapamycin binds the conserved protein FKBP12. The FKBP12-rapamycin complex subsequently binds to TOR1 or TOR2 in TORC1 (mTOR in mTORC1) and thereby inhibits TORC1 signalling. FKBP12-rapamycin fails to bind to TOR2 in yeast TORC2 and thus TORC2 signalling is insensitive to rapamycin 1 . To determine if FKBP12-rapamycin binds to mTORC2, an epitope-tagged version of FKBP12 was precipitated from HEK293 cells treated with drug vehicle or 200 nM rapamycin for 30 min before cell lysis. FKBP12 co-precipitated mTOR and raptor (that is, mTORC1), but

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

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Promiscuous small molecules plague screening libraries and hit lists. Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes. It is not understood how aggregates inhibit their targets. To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregateforming inhibitors with model proteins. By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact. This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein. β-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction. Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates. This association could be reversed or prevented by the addition of Triton X-100. These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition. They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.

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Kinase inhibitors are widely employed as biological reagents and as leads for drug design. Their use is often complicated by their lack of specificity. Although binding conserved ATP sites accounts for some of their nonspecificity, some compounds inhibit proteins not known to bind ATP. It has been found that promiscuous hits from high-throughput screening may act as aggregates. To explore whether this mechanism might explain the action of widely used nonspecific kinase inhibitors, 15 such compounds were studied. Eight of these, rottlerin, quercetin, K-252c, bisindolylmaleimide I, bisindolylmaleimide IX, U0126, indirubin, and indigo, inhibited three diverse non-kinase enzymes. Inhibition was time-dependent and sensitive to enzyme concentration; by light scattering, the compounds formed particles of 100-1000 nm diameter. These observations suggest that these eight kinase inhibitors, at least at micromolar concentrations, are promiscuous and act as aggregates. Results obtained from the use of these compounds at micromolar or higher concentrations against individual enzymes should be interpreted cautiously.

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15–30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP 3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominantnegative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rhodependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as an-tagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic mye-loid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays re-vealed that the serine/threonine kinases Rip-like interact-ing caspase-like apoptosis-regulatory protein kinase (RICK) and p38 were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not dis-criminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be corre-lated with a potent in vivo inhibition of cytokine produc-tion by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods em-ploying immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic

Neuritic extension is the resultant of two vectorial processes: outgrowth and retraction. Whereas myosin IIB is required for neurite outgrowth, retraction is driven by a motor whose identity has remained unknown until now. Preformed neurites in mouse Neuro-2A neuroblastoma cells undergo immediate retraction when exposed to isoform-specific antisense oligonucleotides that suppress myosin IIB expression, ruling out myosin IIB as the retraction motor. When cells were preincubated with antisense oligonucleotides targeting myosin IIA, simultaneous or subsequent addition of myosin IIB antisense oligonucleotides did not elicit neurite retraction, both outgrowth and retraction being curtailed. Even during simultaneous application of antisense oligonucleotides against both myosin isoforms, lamellipodial spreading continued despite the complete inhibition of neurite extension, indicating an uncoupling of lamellipodial dynamics from movement of the neurite. Significantly, lysophosphatidate- or thrombin-induced neurite retraction was blocked not only by the Rho-kinase inhibitor Y27632 but also by antisense oligonucleotides targeting myosin IIA. Control oligonucleotides or antisense oligonucleotides targeting myosin IIB had no effect. In contrast, Y27632 did not inhibit outgrowth, a myosin IIB-dependent process. We conclude that the conventional myosin motor, myosin IIA, drives neurite retraction.