Joseph Avruch

Raptor, a Binding Partner of Target of Rapamycin (TOR), Mediates TOR Action

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.

Sounding the Alarm: Protein Kinase Cascades Activated by Stress and Inflammation

Eukaryotic cells respond to extracellular stimuli by recruiting signal transduction pathways, many of which employ protein Ser/ Thr kinases of the ERK family. The ubiquity of ERKs and their upstream activators, the MEKs, in signal transduction was first appreciated from studies of yeast (1, 2). Although a 54-kDa rat liver c-Jun kinase (SAPK-p54a1) with properties similar to the Rasregulated MAPKs had been characterized (3–5), the physiologic roles and regulation of this and related mammalian enzymes have emerged only recently. Molecular cloning of the SAPKs and p38s, together with the paradigms derived from the “classical” MAPKs and work in lower eukaryotes has enabled rapid elucidation of the regulation and cellular functions of these newer mammalian ERK pathways. Although architecturally homologous to the Ras/MAPK pathway, the SAPK and p38 pathways are not activated primarily by mitogens but by cellular stresses and inflammatory cytokines, which stimuli result in growth arrest, apoptosis, or activation of immune and reticuloendothelial cells.

Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism.

The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p70Δ2–46/ΔCT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase.

Rheb binds and regulates the mTOR kinase

BACKGROUND The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.

Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells

Abstract 1. 1. Plasma membrane and endoplasmic reticulum of free fat cells were isolated in high yield as a mixed microsomal fraction by sucrose gradient centrifugation. 2. 2. The semipermeable behavior of these particles was explored by observing shifts in isopycnic density in continuous polymer (Dextran) gradients over a range of pH and ionic conditions. 3. 3. Using a discontinuous Dextran gradient, a plasma membrane fraction was partially purified. The plasma membrane fraction showed a 12–16-fold enrichment of 5′-nucleotidase over the homogenate. Na + -K + -stimulated ATPase was recovered in highest specific activity in this fraction. Mitochondrial contamination was 3% where endoplasmic reticulum contamination was 27%. 4. 4. A sensitive radioassay for 5′-nucleotidase is described.

Raf meets Ras: completing the framework of a signal transduction pathway

The Ras oncoprotein, a GTP-activated molecular switch, interacts directly with the Raf oncoprotein to recruit the MAP kinases and their subordinates. In this way, a mitogenic signal initiated by tyrosine kinases is converted by Ras into a wave of regulatory phosphorylation on serine and threonine residues that, depending on its intensity and duration, and the variety of substrates available, results in cell differentiation or cell division.

Mammalian MAPK Signal Transduction Pathways Activated by Stress and Inflammation: A 10-Year Update

The mammalian stress-activated families of mitogen-activated protein kinases (MAPKs) were first elucidated in 1994, and by 2001, substantial progress had been made in identifying the architecture of the pathways upstream of these kinases as well as in cataloguing candidate substrates. This information remains largely sound. Nevertheless, an informed understanding of the physiological and pathophysiological roles of these kinases remained to be accomplished. In the past decade, there has been an explosion of new work using RNAi in cells, as well as transgenic, knockout and conditional knockout technology in mice that has provided valuable insight into the functions of stress-activated MAPK pathways. These findings have important implications in our understanding of organ development, innate and acquired immunity, and diseases such as atherosclerosis, tumorigenesis, and type 2 diabetes. These new developments bring us within striking distance of the development and validation of novel treatment strategies. Herein we first summarize the molecular components of the mammalian stress-regulated MAPK pathways and their regulation as described thus far. We then review some of the in vivo functions of these pathways.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro

BACKGROUND The p70 S6 kinase, an enzyme critical for cell-cycle progression through the G1 phase, is activated in vivo by insulin and mitogens through coordinate phosphorylation at multiple sites, regulated by signaling pathways, some of which depend on and some of which are independent of phosphoinositide 3-kinase (Pl 3-kinase). It is not known which protein kinases phosphorylate and activate p70. RESULTS Co-expression of p70 with 3-phosphoinositide-dependent protein kinase 1 (PDK1), a protein kinase that has previously been shown to phosphorylate and activate protein kinase B (PKB, also known as c-Akt), resulted in strong activation of the S6 kinase in vivo. In vitro, PDK1 directly phosphorylated Thr252 in the activation loop of the p70 catalytic domain, the phosphorylation of which is stimulated by PI 3-kinase in vivo and is indispensable for p70 activity. Whereas PDK1-catalyzed phosphorylation and activation of PKB in vitro was highly dependent on the presence of phosphatidylinositol 3,4,5-trisphosphate (Ptdlns (3,4,5)P3), PDK1 catalyzed rapid phosphorylation and activation of p70 in vitro, independent of the presence of Ptdlns(3,4,5)P3. The ability of PDK1 to phosphorylate p70 Thr252 was strongly dependent on the phosphorylation of the p70 noncatalytic carboxy-terminal tail (amino acids 422-525) and of amino acid Thr412. Moreover, once Thr252 was phosphorylated, its ability to cause activation of the p70 S6 kinase was also controlled by the p70 carboxy-terminal tail and by phosphorylation of p70 Ser394, and most importantly, Thr412. The overriding determinant of the absolute p70 activity was the strong positive cooperativity between Thr252 and Thr412 phosphorylation; both sites must be phosphorylated to achieve substantial p70 activation. CONCLUSIONS PDK1 is one of the components of the signaling pathway recruited by Pl 3-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pl 3-kinase.

Mst1 and Mst2 Maintain Hepatocyte Quiescence and Suppress Hepatocellular Carcinoma Development through Inactivation of the Yap1 Oncogene

Hippo-Lats-Yorkie signaling regulates tissue overgrowth and tumorigenesis in Drosophila. We show that the Mst1 and Mst2 protein kinases, the mammalian Hippo orthologs, are cleaved and constitutively activated in the mouse liver. Combined Mst1/2 deficiency in the liver results in loss of inhibitory Ser127 phosphorylation of the Yorkie ortholog, Yap1, massive overgrowth, and hepatocellular carcinoma (HCC). Reexpression of Mst1 in HCC-derived cell lines promotes Yap1 Ser127 phosphorylation and inactivation and abrogates their tumorigenicity. Notably, Mst1/2 inactivates Yap1 in liver through an intermediary kinase distinct from Lats1/2. Approximately 30% of human HCCs show low Yap1(Ser127) phosphorylation and a majority exhibit loss of cleaved, activated Mst1. Mst1/2 inhibition of Yap1 is an important pathway for tumor suppression in liver relevant to human HCC.

Regulation of eIF-4E BP1 Phosphorylation by mTOR

The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/RAFT-1/FRAP is the target through which the rapamycin·FKBP12 complex acts to dephosphorylate/deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin·FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivationin situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner.