Chiharu Tokunaga

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.

A possible linkage between AMP‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway

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.

Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro.

p70 S6 kinase α (p70α) 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 p70α activation in a manner that can be overcome by coexpression of p70α 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 p70α 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 p70α phosphorylation in vitro is accompanied by a substantial restoration in p70α kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70α by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70α 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.

Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that plays a crucial role in a nutrient‐sensitive signalling pathway that regulates cell growth. TOR signalling is potently inhibited by rapamycin, through the direct binding of a FK506‐binding protein 12 (FKBP12)/rapamycin complex to the TOR FRB domain, a segment amino terminal to the kinase catalytic domain. The molecular basis for the inhibitory action of FKBP12/rapamycin remains uncertain. Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that is essential for mTOR signalling in vivo, and whose binding to mTOR is critical for mTOR‐catalysed substrate phosphorylation in vitro. Here we investigated the stability of endogenous mTOR/raptor complex in response to rapamycin in vivo, and to the direct addition of a FKBP12/rapamycin complex in vitro. Rapamycin diminished the recovery of endogenous raptor with endogenous or recombinant mTOR in vivo; this inhibition required the ability of mTOR to bind the FKBP12/rapamycin complex, but was independent of mTOR kinase activity. Rapamycin, in the presence of FKBP12, inhibited the association of raptor with mTOR directly in vitro, and concomitantly reduced the mTOR‐catalysed phosphorylation of raptor‐dependent, but not raptor‐independent substrates; mTOR autophosphorylation was unaltered. These observations indicate that rapamycin inhibits mTOR function, at least in part, by inhibiting the interaction of raptor with mTOR; this action uncouples mTOR from its substrates, and inhibits mTOR signalling without altering mTORs intrinsic catalytic activity.

Protein-Protein Interaction of Zinc Finger LIM Domains with Protein Kinase C

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 associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

Carboxyl‐terminal region conserved among phosphoinositide‐kinase‐related kinases is indispensable for mTOR function in vivo and in vitro

The mammalian target of rapamycin (mTOR) belongs to the family of phosphoinositide (PI)‐kinase‐related kinases that includes the ataxia‐telangiectasia gene product (ATM). mTOR plays a critical role in controlling translational effectors such as p70 S6 kinase α (p70α) and eukaryotic initiation factor 4E binding protein 1 (4EBP1).

Alpha4 protein as a common regulator of type 2A‐related serine/threonine protein phosphatases1

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A‐related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A‐related protein phosphatases.

Kinase Activities Associated with mTOR

Although mTOR is a member of the PI-kinase-related kinase family, mTOR possesses serine-threonine protein kinase activities, which phosphorylate itself and exogenous substrates. mTOR autophosphorylates in vitro and is phosphorylated in vivo on serine residues. Ser2481, which is located in a His-Ser-Phe motif near the conserved carboxyl-terminal mTOR tail, has been reported as an autophosphorylation site in vivo and in vitro. The significance of the autophosphorylation remains unclear. Another phosphorylation site on mTOR in vivo is Ser2448. This site appears not to be an autophosphorylation site but a site potentially phosphorylated by protein kinase B (PKB). mTOR immunopurified from culture cells or tissues phosphorylates in vitro p70 S6 kinase (p70) alpha and p70beta, mainly on Thr412 or Thr401, respectively, located in a Phe-Thr-Tyr motif. Another exogenous substrate phosphorylated by immunopurified mTOR in vitro is eIF4E-binding protein 1 (4E-BP1) at sites corresponding to those phosphorylated in vivo during insulin stimulation in a Ser/Thr-Pro motif. Recently, raptor, a 150-kDa TOR-binding protein that contains a carboxyl-terminal WD-repeat domain, was discovered as a scaffold for the mTOR-catalyzed phosphorylation of 4E-BP1 and for the mTOR-mediated phosphorylation and activation of p70alpha. Other potential substrates phosphorylated by mTOR are nPKCdelta, nPKCepsilon, STAT3, and p53. The requirement of raptor for binding to and phosphorylation by mTOR of these potential substrates would clarify their physiological importance in the mTOR signaling pathway.

Inhibition of amino acid-mTOR signaling by a Leucine derivative induces G1 arrest in Jurkat cells

We have previously demonstrated that N-acetylleucine amide, a derivative of L-leucine, inhibits leucine-induced p70(S6k) activation in a rat hepatoma cell line. In the present study, we investigated whether N-acetylleucine amide is capable of inhibiting amino acid-mTOR signaling. N-Acetylleucine amide caused cell cycle arrest at G1 stage in Jurkat cells, a human leukemia T cell line, concomitant with the inhibition of serum-induced p70(S6k) activation and p27 degradation. Treatment of Jurkat cells with this compound also exhibited dephosphorylation of retinoblastoma protein. These effects are similar to the inhibitory effects of rapamycin on amino acid-mTOR signaling pathway and suggest that N-acetylleucine amide acts as a rapamycin-like reagent to inhibit cell cycle progression in Jurkat cells.

Distinct regulatory mechanism for p70 S6 kinase beta from that for p70 S6 kinase alpha.

Background A novel ribosomal S6 kinase, termed p70 S6 kinase β (p70β), has a highly homologous amino acid sequence to that of p70/p85 S6 kinase (p70α). This includes the critical phosphorylation sites, Thr252, Ser394 and Thr412 in p70α1, which correspond to Thr241, Ser383 and Thr401 in p70β1, respectively. However, the regulatory mechanism for p70β remains to be elucidated.