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.

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid-and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.The target of rapamycin (TOR) 1 proteins are protein kinases that were first identified in Saccharomyces cerevisiae through mutants that confer resistance to growth inhibition induced by the immunosuppressive macrolide rapamycin (1). In mammalian cells, rapamycin blocks phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (2, 3) and p70 S6 kinase (p70S6k) (4,5) by interfering with the function of mTOR (6, 7) (also known as FRAP, RAFT1, or RAPT). Although mTOR can phosphorylate both these targets directly in vitro (8 -10), the mechanism of mTOR regulation of these phosphorylations in vivo remains incompletely understood (11).The p70S6k is activated through a sequential multisite phosphorylation in response to insulin or mitogens in vivo (11). In addition, nutrients, especially amino acids, have been shown to regulate the phosphorylation of p70S6k and 4E-BP1 and to be necessary for insulin or mitogen regulation (12-17). The activity of p70S6k␣1 in vivo is most closely related to the phosphorylation at Thr-412, situated in a hydrophobic motif C-terminal to the canonical catalytic domain (18,19). The identity of the kinase(s) acting on this site in vivo is uncertain; however, this site can be phosphorylated directly by mTOR in vitro (9, 10). Recently, site-specific mutagenesis was employed to define a five-amino acid sequence called the TOS (TOR signaling) motif as the minimal functionally important region within this p70S6k noncatalytic N-terminal segment (21). As with N-terminal deletion, mutation of a single Phe within the TOS motif to Ala causes marked inhibition of activity of full-length p70S6k and a loss of sensitivity to rapamycin and amino acid withdrawal in the p70S6k-⌬CT104, lacking C-terminal noncatalytic tail, background. In addition, a TOS motif was identified in the 4E-BPs, wherein mutation of 4E-BP1 Phe-114 to Ala inhibits amino acid-and serum-induced 4E-BP1 phosphorylation.Raptor is a recently...

p70 S6 kinase ␣ (p70␣) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70␣ activation in a manner that can be overcome by coexpression of p70␣ with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70␣ polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70␣ phosphorylation in vitro is accompanied by a substantial restoration in p70␣ kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70␣ by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70␣ activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase ␣ (p70␣), 1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site (1). p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues (2). Several sets of independently regulated p70␣ phosphorylation sites have been identified (3-6); one set consists of Ser/Thr-Pro motifs, five of which are clustered in a psuedosubstrate autoinhibitory domain in the noncatalytic carboxyl-terminal tail (Ser-434, Ser-441, Ser-447, Ser-452, and Thr-444 in p70␣), and two others, Thr-390 and Ser-394, are located in a 65-amino acid segment immediately carboxyl-terminal to the kinase catalytic domain. A second set of regulated phosphorylation sites, Thr-412 and Ser-427, exhibit the sequence motif Phe-Ser/Thr-Phe/Tyr. Thr-252, located on the activation loop in catalytic subdomain VIII, is the site at which 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates p70␣ (7,8). Among these, the phosphorylation of Thr-252, Ser-394, and Thr-412 is necessary for the activation of p70␣ kinase catalytic function; the attainment of physiologic levels of p70␣ activity results from a strongly synergistic, positive site-site interaction between the phosphorylated Thr-252 and Thr-412 residues (7).In addition to its regulation by insulin and mitogens through PI-3 kinase-dependent pathways, p70␣ can also be activated by increasing concentrations of extracellular amino acids in the absence of serum or mitogens to the level attained by maximal mitogen stimulation (9-11). Moreover, a thr...

Background : The mammalian target of rapamycin (mTOR) regulates multiple cellular functions including translation in response to nutrients, especially amino acids. AMP-activated protein kinase (AMPK) modulates metabolism in response to energy demand by responding to changes in AMP.

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to as-The LIM domain is a Cys-rich domain composed of 50 -60 amino acid residues with the consensus sequence (Cys-X 2 -Cys-X 17-19 -His-X 2 -Cys) -X 2 -(Cys-X 2 -Cys-X 16 -20 -Cys-X 2 -His/Asp /Cys) (where X represents any amino acid) and is found in various proteins (1, 2): homeodomain-containing transcription factors, cytoskeletal proteins, LIM domain only proteins, protein kinases, and proteins of undefined function. Physicochemical and structural analyses have revealed that the LIM domain is composed of two independent zinc-coordinated fingers (3, 4). Although many zinc finger motifs bind to specific DNA or RNA sequences (5), the LIM domain has been proposed to participate in protein-protein interactions (1, 2). In fact, five proteins have been reported recently as a highly specific target of each LIM domain: the LIM1 domain of zyxin binds to the LIM-only protein CRP by LIM-LIM interaction (6); the CRP forms homodimer by LIM-LIM interaction (7); the LIM domain of RBTN2 binds to the bHLH (basichelix-loop-helix) domain of TAL1 protein (8); the LIM2 and LIM3 domains of Enigma interact with the Tyr-containing tight-turn motifs of the GDNF 1 receptor (GDNFR, known as a Ret Tyr kinase) and the insulin receptor (InsR), respectively (9, 10). Although most LIM domains adopt a similar zinc-coordinated finger consisting of well conserved amino acid sequences, no protein has been identified yet as a common target of LIM domains.The PKC family consists of at least 11 isoforms, which play distinct roles for many cellular functions but show subtle difference of substrate specificities by in vitro phosphorylation studies (11,12). Therefore, it is reasonable to assume that there are some mechanisms by which each PKC isoform recognizes its specific substrate proteins in vivo. Recently, several proteins associating with PKC have been emerged to govern the subcellular localization of the enzyme family (13-16).We report here a novel PKC-binding protein containing three LIM domains, designated ENH, and show the association of PKC with LIM domains of different proteins including this novel PKC-binding protein, suggesting that protein-protein interaction with PKC is a general property of LIM domains. EXPERIMENTAL PROCEDURESYeast Two-Hybrid Assay-The yeast two-hybrid assay (17) was conducted in the yeast strain CG-1945 , a derivative of HF7c (18), by using a fusion between GAL4 DNA binding domain and the regulatory domain of rat PKC ␤I (residues 1-340) (19) as a bait. ␤-Galactosidase activity in yeast cells was measured by plate assay methods. All measurements were repeated at least four times.Expression of Epitope-tagged ENH in COS-7 Cells-We constructed two parental vectors, pTB701-FLAG and pTB701-HA, for expression o...