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Objective: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific rictor knockout (FRic(-/-)) mice., Research Design and Methods: Insulin signaling and glucose and lipid metabolism were studied in FRic(-/-) fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp., Results: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected. The signaling defects in FRic(-/-) fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, rictor-null fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole-body level of FRic(-/-) mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis., Conclusions: Rictor/mTORC2 in fat cells plays an important role in whole-body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.

Lipin 1 is a bifunctional protein that regulates gene transcription and, as a Mg(2+)-dependent phosphatidic acid phosphatase (PAP), is a key enzyme in the biosynthesis of phospholipids and triacylglycerol. We describe here the functional interaction between lipin 1 and the nuclear factor of activated T cells c4 (NFATc4). Lipin 1 represses NFATc4 transcriptional activity through protein-protein interaction, and lipin 1 is present at the promoters of NFATc4 transcriptional targets in vivo. Catalytically active and inactive lipin 1 can suppress NFATc4 transcriptional activity, and this suppression may involve recruitment of histone deacetylases to target promoters. In fat pads from mice deficient for lipin 1 (fld mice) and in 3T3-L1 adipocytes depleted of lipin 1 there is increased expression of several NFAT target genes including tumor necrosis factor alpha, resistin, FABP4, and PPARgamma. Finally, both lipin 1 protein and total PAP activity are decreased with increasing adiposity in the visceral, but not subcutaneous, fat pads of ob/ob mice. These observations place lipin 1 as a potentially important link between triacylglycerol synthesis and adipose tissue inflammation.

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser(863), reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser(863) mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.

Objective: Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches., Methods and Results: Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1-deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling., Conclusions: Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.

A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contains the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus [Satow R, Chan TC, Asashima M (2002) Biochem Biophys Res Commun 295:85-91]. Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains and show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1Delta yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p [Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S (2005) EMBO J 24:1931-1941], and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis, and that this cascade is conserved from yeast to mammals.

Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

Perturbations in hepatic lipid homeostasis are linked to the development of obesity-related steatohepatitis. Mutations in the gene encoding lipin 1 cause hepatic steatosis in fld mice, a genetic model of lipodystrophy. However, the molecular function of lipin 1 is unclear. Herein, we demonstrate that the expression of lipin 1 is induced by peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a transcriptional coactivator controlling several key hepatic metabolic pathways. Gain-of-function and loss-of-function strategies demonstrated that lipin selectively activates a subset of PGC-1alpha target pathways, including fatty acid oxidation and mitochondrial oxidative phosphorylation, while suppressing the lipogenic program and lowering circulating lipid levels. Lipin activates mitochondrial fatty acid oxidative metabolism by inducing expression of the nuclear receptor PPARalpha, a known PGC-1alpha target, and via direct physical interactions with PPARalpha and PGC-1alpha. These results identify lipin 1 as a selective physiological amplifier of the PGC-1alpha/PPARalpha-mediated control of hepatic lipid metabolism.

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.

Insulin stimulates protein synthesis by increasing translation initiation. This response is mediated by mTOR and is believed to result from 4EBP1 phosphorylation, which allows eIF4E to bind eIF4G. Here, we present evidence that mTOR interacts directly with eIF3 and that mTOR controls the association of eIF3 and eIF4G. Activating mTOR signaling with insulin increased by as much as five-fold the amount of eIF4G bound to eIF3. This novel effect was blocked by rapamycin and other inhibitors of mTOR, and it required neither eIF4E binding to eIF4G nor eIF3 binding to the 40S ribosomal subunit. The increase in eIF4G associated with eIF3 occurred rapidly and at physiological concentrations of insulin. Moreover, the magnitude of the response was similar to the increase in eIF4E binding to eIF4G produced by insulin. Thus, increasing eIF4G association with eIF3 represents a potentially important mechanism by which insulin, as well as amino acids and growth factors that activate mTOR, stimulate translation.

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.

The mammalian target of rapamycin (mTOR) functions with raptor and mLST8 in a signaling complex that controls rates of cell growth and proliferation. Recent results indicate that an inhibitor of the Ras signaling pathway, farnesylthiosalicylic acid (FTS), decreased phosphorylation of the mTOR effectors, PHAS-I and S6K1, in breast cancer cells. Here we show that incubating 293T cells with FTS produced a stable change in mTOR activity that could be measured in immune complex kinase assays using purified PHAS-I as substrate. Similarly, FTS decreased the PHAS-I kinase activity of mTOR when added to cell extracts or to immune complexes containing mTOR. Incubating either cells or extracts with FTS also decreased the amount of raptor that coimmunoprecipitated with mTOR, although having relatively little effect on the amount of mLST8 that coimmunoprecipitated. The concentration effect curves of FTS for inhibition of mTOR activity and for dissociation of the raptor-mTOR complex were almost identical. Caffeine, wortmannin, LY294002, and rapamycin-FKBP12 also markedly inhibited mTOR activity in vitro, but unlike FTS, none of the other mTOR inhibitors appreciably changed the amount of raptor associated with mTOR. Thus, our findings indicate that FTS represents a new type of mTOR inhibitor, which acts by dissociating the functional mTOR-raptor signaling complex.

Amino acids are nutrients responsible for mammalian target of rapamycin (mTOR) regulation in mammalian cells. The mTOR protein is mainly known for its role in regulating cell growth, notably via protein synthesis. In addition to amino acids, mTOR is regulated by insulin via a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. mTOR mediates crosstalk between amino acids and insulin signaling. We show that in freshly isolated rat adipocytes, insulin stimulates the phosphorylation of mTOR on serine 2448, a protein kinase B (PKB) consensus phosphorylation site. This site is also phosphorylated by amino acids, which in contrast to insulin do not activate PKB. Moreover, insulin and amino acids have an additive effect on mTOR phosphorylation, indicating that they act via two independent pathways. Importantly, amino acids, notably leucine, permit insulin to stimulate PKB when PI 3-kinase is inhibited. They also rescue glucose transport and the mTOR pathway. Further, leucine alone can improve insulin activation of PKB in db/db mice. Our results define the importance of amino acids in insulin signaling and reveal leucine as a key amino acid in disease situations associated with insulin-resistance in adipocytes.

The major function of mammalian target of rapamycin (mTOR) is the control of cell growth. Insulin and amino acids regulate the mTOR pathway, and both are needed to promote its maximal activation. To further understand mTOR regulation by insulin and amino acids, we have studied the enzyme in primary cultures of hepatocytes. We show that insulin increases mTOR phosphorylation on Ser2448, a consensus phosphorylation site for protein kinase B (PKB). Ser2448 phosphorylation is also increased by amino acids, although they do not activate PKB. Furthermore, insulin and amino acids have an additive effect, indicating that they act through distinct pathways. We also show that phosphorylation of Ser2448 does not seem to modulate in vitro phosphorylation of eukaryotic initiation factor 4E-binding protein 1 by mTOR. However, stimulation of hepatocytes with insulin and amino acids leads to an increase in mTOR kinase activity. Rapamycin has no effect on insulin-, glucagon-, and 8-(4-chlorophenylthio)adenosine-cAMP-induced amino acid transport. Surprisingly, glucagon and 8-(4-chlorophenylthio)adenosine-cAMP, which do not activate PKB, stimulate the phosphorylation on Ser2448 of mTOR. However, glucagon inhibits amino acid- and insulin-induced activation of ribosomal S6 protein kinase 1 and phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1. Our results demonstrate that glucagon, which is not able to activate but rather inhibits the mTOR pathways, stimulates the phosphorylation of mTOR on Ser2448. This finding suggests that phosphorylation of this site might not be sufficient for mTOR kinase activity but is likely to be involved in other functions.

Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in noncontracted and postcontracted muscle of GSL3-transgenic and wild-type mice. GSL3-transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3-transgenic and wild-type mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty minutes of contraction significantly reduced muscle glycogen concentration in wild-type (49%) and transgenic (27%) mice, although transgenic mice retained 168.8 +/- 20.5 micromol/g glycogen compared with 17.7 +/- 2.6 micromol/g glycogen for wild-type mice. Muscle of transgenic and wild-type mice demonstrated similar pre- (3.6 +/- 0.3 and 3.9 +/- 0.6 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) and postcontraction (7.9 +/- 0.4 and 7.0 +/- 0.4 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) insulin-stimulated glucose uptakes. However, the [14C]glucose incorporated into glycogen was greater in noncontracted (151%) and postcontracted (157%) transgenic muscle vs. muscle of corresponding wild-type mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.

The mammalian target of rapamycin, mTOR, is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. The activity of mTOR is controlled not only by amino acids, but also by hormones and growth factors that activate the protein kinase Akt. The signaling pathway downstream of Akt leading to mTOR involves the protein products of the genes mutated in tuberous sclerosis, TSC1 and TSC2, and the small guanosine triphosphatase, Rheb. In cells, mTOR is found in a complex with two other proteins, raptor and mLST8. In this review, we describe recent progress in understanding the control of the mTOR signaling pathway and the role of mTOR-interacting proteins.

Insulin stimulates phosphorylation of multiple sites in the eIF4E-binding protein, PHAS-I, leading to dissociation of the PHAS-I.eIF4E complex and to an increase in cap-dependent translation. The Ser-64 and Ser-111 sites have been proposed to have key roles in controlling the association of PHAS-I and eIF4E. To determine whether the effects of insulin require these sites, we assessed the control of PHAS-I proteins having Ala-64 or Ala-111 mutations. The results indicate that phosphorylation of neither site is required for insulin to promote release of PHAS-I from eIF4E. Also, the mutation of Ser-111, which has been proposed to serve as a necessary priming site for the phosphorylation of other sites in PHAS-I, did not affect the phosphorylation of Thr-36/45, Ser-64, or Thr-69. Insulin promoted the release of eIF4E from PHAS-II, a PHAS isoform that lacks the Ser-111 site, but it was without effect on the amount of eIF4E bound to the third isoform, PHAS-III. The results demonstrate that contrary to widely accepted models, Ser-64 and Ser-111 are not required for the control of PHAS-I binding to eIF4E in cells, implicating phosphorylation of the Thr sites in dissociation of the PHAS-I.eIF4E complex. The findings also indicate that PHAS-II, but not PHAS-III, contributes to the control of protein synthesis by insulin.

Mammalian target of rapamycin (mTOR) is the central element of a signaling pathway involved in the control of mRNA translation and cell growth. The actions of mTOR are mediated in part through the phosphorylation of the eukaryotic initiation factor 4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eukaryotic initiation factor 4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The Arg-Ala-Ile-Pro (RAIP motif) and Phe-Glu-Met-Asp-Ile (tor signaling motif) sequences found in the NH2- and COOH-terminal regions of PHAS-I, respectively, are required for the efficient phosphorylation of PHAS-I in cells. Here we show that mutations in either motif markedly decreased the phosphorylation of recombinant PHAS-I by mTOR in vitro. Wild-type PHAS-I, but none of the mutant proteins, was coimmunoprecipitated with hemagglutinin-tagged raptor, an mTOR-associated protein, after extracts of cells overexpressing raptor had been supplemented with recombinant PHAS-I proteins. Moreover, raptor overexpression enhanced the phosphorylation of wild-type PHAS-I by mTOR but not the phosphorylation of the mutant proteins. The results not only provide direct evidence that both the RAIP and tor signaling motifs are important for the phosphorylation by mTOR, possibly by allowing PHAS-I binding to raptor, but also support the view that mTOR phosphorylates PHAS-I in cells.

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.

We have investigated the effects of insulin, amino acids, and the degree of muscle loading on the phosphorylation of Ser(2448), a site in the mammalian target of rapamycin (mTOR) phosphorylated by protein kinase B (PKB) in vitro. Phosphorylation was assessed by immunoblotting with a phosphospecific antibody (anti-Ser(P)(2448)) and with mTAb1, an activating antibody whose binding is inhibited by phosphorylation in the region of mTOR that contains Ser(2448). Incubating rat diaphragm muscles with insulin increased Ser(2448) phosphorylation but did not change the total amount of mTOR. Insulin, but not amino acids, activated PKB, as evidenced by increased phosphorylation of both Ser(308) and Thr(473) in the kinase. Ser(2448) phosphorylation was also modulated by muscle-loading. Overloading the rat plantaris muscle by synergist muscle ablation, which promotes hypertrophy of the plantaris muscle, increased Ser(2448) phosphorylation. In contrast, unloading the gastrocnemius muscle by hindlimb suspension, which promotes atrophy of the muscle, decreased Ser(2448) phosphorylation, an effect that was fully reversible. Neither overloading nor hindlimb suspension significantly changed the total amount of mTOR. In summary, our results demonstrate that atrophy and hypertrophy of skeletal muscle are associated with decreases and increases in Ser(2448) phosphorylation, suggesting that modulation of this site may have an important role in the control of protein synthesis.

The phosphorylation of a previously uncharacterized protein of apparent M(r) approximately 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy (fld) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121-124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent M(r) from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.

Muscle mass is influenced by many factors including genetically programmed changes, hormonal state, level of activity, and disease processes. Ultimately, whether or not a muscle hypertrophies or atrophies is determined by a simple relationship between the rates of protein synthesis and degradation. When synthesis exceeds degradation, the muscle hypertrophies, and vice versa. In contrast to this simple relationship, the processes that control muscle protein synthesis and degradation are complex. Recently, significant progress has been made in understanding the biochemical mechanisms that control the rate of translation initiation, which is generally the limiting phase in protein synthesis.

Background: The authors previously reported the generation of a knockout mouse model of Pompe disease caused by the inherited deficiency of lysosomal acid alpha-glucosidase (GAA). The disorder in the knockout mice (GAA-/-) resembles the human disease closely, except that the clinical symptoms develop late relative to the lifespan of the animals. In an attempt to accelerate the course of the disease in the knockouts, the authors increased the level of cytoplasmic glycogen by overexpressing glycogen synthase (GSase) or GlutI glucose transporter., Methods: GAA-/- mice were crossed to transgenic mice overexpressing GSase or GlutI in skeletal muscle., Results: Both transgenics on a GAA knockout background (GS/GAA-/- and GlutI/GAA-/-) developed a severe muscle wasting disorder with an early age at onset. This finding, however, is not the major focus of the study. Unexpectedly, the mice bearing the GSase transgene, but not those bearing the GlutI transgene, accumulated structurally abnormal polysaccharide (polyglucosan) similar to that observed in patients with Lafora disease, glycogenosis type IV, and glycogenosis type VII. Ultrastructurally, the periodic acid-Schiff (PAS)-positive polysaccharide inclusions were composed of short, amorphous, irregular branching filaments indistinguishable from classic polyglucosan bodies. The authors show here that increased level of GSase in the presence of normal glycogen branching enzyme (GBE) activity leads to polyglucosan accumulation. The authors have further shown that inactivation of lysosomal acid alpha-glucosidase in the knockout mice does not contribute to the process of polyglucosan formation., Conclusions: An imbalance between GSase and GBE activities is proposed as the mechanism involved in the production of polyglucosan bodies. The authors may have inadvertently created a "muscle polyglucosan disease" by simulating the mechanism for polyglucosan formation.

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.

In diabetic patients, alpha-lipoic acid (LA) improves skeletal muscle glucose transport, resulting in increased glucose disposal; however, the molecular mechanism of action of LA is presently unknown. We studied the effects of LA on basal and insulin-stimulated glucose transport in cultured rat L6 muscle cells that overexpress GLUT4. When 2-deoxy-D-glucose uptake was measured in these cells, they were more sensitive and responsive to insulin than wild-type L6 cells. LA, at concentrations < or = 1 mmol/l, had only small effects on glucose transport in cells not exposed to oxidative stress. When cells were exposed to glucose oxidase and glucose to generate H2O2 and cause oxidative stress, there was a marked decrease in insulin-stimulated glucose transport. Pretreatment with LA over the concentration range of 10-1,000 pmol/l protected the insulin effect from inhibition by H2O2. Both the R and S isomers of LA were equally effective. In addition, oxidative stress caused a significant decrease (approximately 50%) in reduced glutathione concentration, along with the rapid activation of the stress-sensitive p38 mitogen-activated protein kinase. Pretreatment with LA prevented both of these events, coincident with protecting insulin action. These studies indicate that in muscle, the major site of insulin-stimulated glucose disposal, one important effect of LA on the insulin-signaling cascade is to protect cells from oxidative stress-induced insulin resistance.

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.

Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.

Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio)adenosine 3'-5'-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin. These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70(S6K), and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR.

Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70(S6k), activated downstream of mTOR, was similar in four cell lines (range, 11.75-41. 8 pmol/2 x 10(6) cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed >/=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.

Amino acids have been identified as important signaling molecules involved in pancreatic beta-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, alpha-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by beta-cells, we investigated whether endogenous insulin secreted by beta-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 microM) and herbimycin A (1 microM), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from beta-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nM) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70(s6) kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other beta-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote beta-cell proliferation either by stimulating phosphorylation of PHAS-I and p70(s6k) via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.

The primary site in PHAS-I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS-I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS-I to eIF4E, and results of peptide mapping experiments with PHAS-I immunoprecipitated from 32P-labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS-I.

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

Although glucose regulates the biosynthesis of a variety of beta cell proteins at the level of translation, the mechanism responsible for this effect is unknown. We demonstrate that incubation of pancreatic islets with elevated glucose levels results in rapid and concentration-dependent phosphorylation of PHAS-I, an inhibitor of mRNA cap-binding protein, eukaryotic initiation factor (eIF)-4E. Our initial approach was to determine if this effect is mediated by the metabolism of glucose and activation of islet cell protein kinases, or whether insulin secreted from the beta cell stimulates phosphorylation of PHAS-I via an insulin-receptor mechanism as described for insulin-sensitive cells. In support of the latter mechanism, inhibitors of islet cell protein kinases A and C exert no effect on glucose-stimulated phosphorylation of PHAS-I, whereas the phosphatidylinositol 3-kinase inhibitor, wortmannin, the immunosuppressant, rapamycin, and theophylline, a phosphodiesterase inhibitor, promote marked dephosphorylation of PHAS-I. In addition, exogenous insulin and endogenous insulin secreted by the beta cell line, betaTC6-F7, increase phosphorylation of PHAS-I, suggesting that beta cells of the islet, in part, mediate this effect. Studies with beta cell lines and islets indicate that amino acids are required for glucose or exogenous insulin to stimulate the phosphorylation of PHAS-I, and amino acids alone dose-dependently stimulate the phosphorylation of PHAS-I, which is further enhanced by insulin. Furthermore, rapamycin inhibits by approximately 62% the increase in total protein synthesis stimulated by high glucose concentrations. These results indicate that glucose stimulates PHAS-I phosphorylation via insulin interacting with its own receptor on the beta cell which may serve as an important mechanism for autoregulation of protein synthesis by translation.

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

PHAS-I is the prototype of a group of eIF4E-binding proteins that can regulate mRNA translation in response to hormones and growth factors. To investigate the importance of PHAS-I in the physiology of the intact animal, we disrupted the PHAS-I gene in mice. Tissues and cells derived from the knockout mice contained no detectable PHAS-I protein. A related protein, PHAS-II, and eIF4E were readily detectable in tissues from these animals, but neither appeared to be changed in a compensatory manner. Mice lacking PHAS-I appeared normal at birth. However, male knockout mice weighed approximately 10% less than controls at all ages, whereas female weights were similar to those of controls. Both males and females were fertile. Tissues from adult animals appeared to be normal by routine histological staining techniques, as were routine blood cell counts and chemistries. Fibroblasts derived from PHAS-I-deficient mouse embryos exhibited normal rates of growth and overall protein synthesis, responded normally to serum stimulation of ornithine decarboxylase activity and cell growth, and rapamycin inhibition of cell growth. Under these experimental conditions, PHAS-I is apparently not required for the normal development and reproductive behavior of female mice, but is required for normal body weight in male mice; the mechanisms responsible for this phenotype remain to be determined.

Insulin and growth factors elicit rapid increases in protein synthesis by stimulating mRNA translation. PHAS/4E-BPs, a recently discovered family of elF4E-binding, proteins, appear to play a key role in this process, as well as in the control of cell proliferation.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

PHAS-I is a recently discovered regulator of translation initiation. Non-phosphorylated PHAS-I binds and inhibits eukaryotic initiation factor-4E, the mRNA cap-binding protein that mediates a rate-limiting step in translation initiation. When PHAS-I is phosphorylated in response to insulin, the PHAS-I/eukaryotic initiation factor-4E complex dissociates. The present study was conducted to investigate mechanisms involved in the control of PHAS-I. Phosphorylation of PHAS-I was monitored by immunoblotting after subjecting extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This was possible because phosphorylation markedly decreases the electrophoretic mobility of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortmannin inhibited insulin-stimulated phosphorylation of PHAS-I at concentrations similar to those that inhibited activation of p70S6K. Both agents increased the amount of PHAS-I that co-purified with eukaryotic initiation factor-4E when extracts were fractionated using a cap affinity resin, indicating that PHAS-I binding to the initiation factor was increased. Incubating adipocytes with the protein phosphatase inhibitors, calyculin A and okadaic acid, increased PHAS-I phosphorylation and opposed the effects of rapamycin on decreasing PHAS-I phosphorylation. However, neither okadaic acid nor calyculin A abolished the effects of rapamycin on PHAS-I. These results suggest that the phosphorylation of PHAS-I in response to insulin occurs via the p70S6K signalling pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may have important roles in the control of both protein synthesis and mitogenesis.

Incubating either Chinese hamster ovary (CHO) cells or 3T3-L1 adipocytes with insulin increased the phosphorylation of the eIF-4E-binding protein, PHAS-I. Insulin also activated p70S6K and the Erk-1 and Erk-2 isoforms of mitogen-activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10-100 times higher than those needed to increase PHAS-I phosphorylation and p70S6K activity. Incubating cells with the inhibitor of MAP kinase kinase (MEK) activation, PD 098059, blocked the effects of low concentrations of insulin on PHAS-I and p70S6K. The effects of the inhibitor were overcome by increasing concentrations of insulin. The results indicate that insulin activates a PD 098059-sensitive kinase that is involved in the regulation of both p70S6K and PHAS-I.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

The metabolism of the storage polysaccharide glycogen is intimately linked with insulin action and blood glucose homeostasis. Insulin activates both glucose transport and glycogen synthase in skeletal muscle. The central issue of a long-standing debate is which of these two effects determines the rate of glycogen synthesis in response to insulin. Recent studies with transgenic animals indicate that, under appropriate conditions, each process can contribute to determining the extent of glycogen accumulation. Insulin causes stable activation of glycogen synthase by promoting dephosphorylation of multiple sites in the enzyme. A model linking this action to the mitogen-activated protein kinase signaling pathway via the phosphorylation of the regulatory subunit of glycogen synthase phosphatase gained widespread acceptance. However, the most recent evidence argues strongly against this mechanism. A newer model, in which insulin inactivates the enzyme glycogen synthase kinase-3 via the protein kinase B pathway, has emerged. Though promising, this model still does not completely explain the molecular basis for the insulin-mediated activation of glycogen synthase, which remains one of the many unknowns of insulin action.

Insulin stimulated protein synthesis in gastrocnemius muscle of perfused rat hindlimb preparations by approximately twofold. The stimulation of protein synthesis was associated with a 12-fold increase in the amount of eukaryotic initiation factor eIF-4G bound to the mRNA cap-binding protein eIF-4E. In part, the increased binding of eIF-4G to eIF-4E was a result of release of eIF-4E bound to the translational regulator, PHAS-I, through a mechanism involving enhanced phosphorylation of PHAS-I. However, the insulin-induced association of eIF-4E and eIF-4G was not due to increased net phosphorylation of eIF-4E because insulin decreased the amount present in the phosphorylated form from 86 to 59% of total eIF-4E. Overall, the results suggest that insulin stimulates protein synthesis in gastrocnemius muscle through a mechanism involving increased binding of eIF-4G to eIF-4E, which is in part due to phosphorylation of PHAS-I, resulting in a release of eIF-4E from the inactive PHAS-I x eIF-4E complex.