rapamycin , an antifungal macrolide purified from streptomyces hygroscopicus ( abraham & wiederrecht , 1996 ) has potent immunosuppressant and antiproliferative properties and currently is used as a fda - approved immunosuppressant and anticancer drug . its cellular target was identified from yeast genetic screening in which mutation of tor1 - 1 and tor2 - 1 genes showed resistance to the growth - inhibitory properties of rapamycin ( heitman et al . , 1991 ) . tor homologs have also been identified in plants ( attor in arabidopsis thaliana ) ( menand et al . , 2002 ) , fungus ( tor1 in cryptcococcus neoformans ) ( cruz et al . , 1999 ) , caenorhabditis elegans ( cetor ) ( hara et al . , 2002 ) , drosophila ( dtor ) ( oldham et al . , 2000 ; zhang et al . , 2003 ) , and mammals ( mtor ) ( brown et al . , 1994 ; chiu et al . , 1994 ; tor is a serine / threonine protein kinase and a member of phosphatidylinositol kinase - related kinase ( pikk ) family ( schmelzle & hall , 2000 ) . it consists of 20 tandem heat repeats at the n - terminal followed by an fat and frb ( fkbp12/rapamycin binding ) domains . mtor kinase domain is located in between frb and fatc ( fat c - terminus ) domain at the c - terminus of the protein ( inoki et al . , 2005a ) . rapamycin binds with immunophilin fkbp12 ( fk506-binding protein 12 kda ) in the cell and forms a complex ( abraham & wiederrecht , 1996 ) . it appears that this fkbp12-rapamycin complex binds to frb domain and inhibits physiological functions of mtor , however , exact mechanism has not been elucidated yet . mtor exists in two distinct protein complexes , mtor complex1 ( mtorc1 ) and mtor complex2 ( mtorc2 ) ( hara et al . , 2002 ; kim et al . , 2002 ; jacinto et al mtorc1 consists of mtor , raptor , mlst8 ( gl ) , pras40 ( proline - rich akt / pkb substrate 40 kda ) , and recently identified fkbp38 ( bai et al . mtorc1 regulates the rate of protein synthesis and cell growth in a rapamycin sensitive way ( fig . 1 ) ( fingar et al . , 2002 ; hay & sonenberg , 2004 ) . while in mtorc2 , mtor interacts with rictor , mlst8 , protor ( protein observed with rictor ) ( pearce et al . , 2007 ) , and msin1 ( reviewed in ( yang & guan , 2007 ) ) . unlike mtorc1 , mtorc2 activity is not inhibited by rapamycin at least in a short time period ( jacinto et al . , 2004 ; sarbassov et al . , substrates of mtorc2 include akt and sgk ( serum and glucocorticoid - inducible kinase ) ( garcia - martinez & alessi , 2008 ) . 2008 ) and has known to be involved in cytoskeletal organization ( loewith et al . , 2002 ; jacinto et al . , 2004 ) . raptor is a scaffold protein which recruits substrates to mtor and knock - down of raptor abolishes physiological activity of mtor ( hara et al . , 2002 ; kim et al . , 2002 ) mlst8 binds to mtor kinase domain and activates the kinase activity independent of raptor ( kim et al . recent in vivo studies using mlst8-/- mefs ( mouse embryonic fibroblasts ) , however , showed that an ability of mtor to phosphorylate its substrates , s6k and 4ebp1 , and to interact with raptor was not impaired in these cells ( guertin et al . , 2006 ) , which suggests that mlst8 may not be an indispensable component of mtorc1 function . pras40 acts as a negative regulator of mtorc1 either by binding directly to the mtor kinase domain and inhibits kinase activity ( vander haar et al . , 2007 ) or by association with raptor via a tor signaling motif ( tos motif ) in pras40 , which can cause substrate competition to raptor ( oshiro et al . , 2007 on the other hand , insulin stimulation phosphorylates thr246 site of pras40 and relieves its inhibitory effect on mtorc1 , which suggests that pras40 mediates growth factor signals to mtorc1 . ( 2007 ) identified that fkbp38 also acts as a negative regulator of mtorc1 and overexpression of fkbp38 inhibits s6k1 ( t389 ) , s6 ( s235/236 ) , and 4ebp1 ( t37/46 ) phosphorylation . mtorc1 recruits and phosphorylates s6k1 and 4ebp1 via interaction of raptor and tos motif located at the n - terminus and c - terminus of s6k1 and 4ebp1 , respectively ( reviewed in ( fingar et al . , 2002 ; hay & sonenberg , 2004 ) ) . activated s6k1 phosphorylates 40s ribosomal protein s6 , which has been suggested to selectively increase the translation of subset of mrnas containing a terminal oligopyrimidine ( top ) tract at their 5'-end . since many of the 5'top mrnas encode ribosomal proteins and other translation factors , activated s6k by mtor is thought to increase ribosome biogenesis . its unphosphorylated form binds and inhibits eukaryotic translation initiation factor 4e ( eif4e ) which binds to the cap structure ( mgpppn ) at the 5'-end of mrna transcripts . phosphorylation of 4ebp1 by mtor dissociates tight 4ebp1 binding from eif4e from which eif4e recruits other components of the translation initiation complex and initiates cap - dependent translation , which increases overall translational capacity of the cell . s6k1 also phosphorylates translation inactivating kinase , eukaryotic elongation factor2 ( eef2 ) kinase , and inactivates its function ( redpath et al . , 1996 ) . the activity of mtorc1 is finely tuned by upstream positive and negative regulators ( hay & sonenberg , 2004 ) . rheb ( ras - homolog enriched in brain ) small gtpase is a positive regulator of mtorc1 and is indispensable for mtor activity . it directly binds to the carboxyterminal lobe of the mtor catalytic domain and modulates the kinase activity ( long et al . , 2005a s6k1 and 4ebp1 can not be phosphorylated if rheb gene is knocked down ( zhang et al . , 2003 ) , whereas , overexpression of rheb stimulates s6k1 and 4ebp1 phosphorylation ( inoki et al . , 2003a ; tee et al . , 2003 ) tsc ( tuberous sclerosis complex ) 1 and 2 heterodimer acts as rheb gap ( gtpase activation protein ) and converts rheb to gdp - bound inactive form ( garami et al . , 2003 ; inoki et al . , 2003a ; tee et al . , 2003 ) . tsc1/2 integrates a variety of signals including insulin , growth factors , and energy status and negatively regulates mtorc1 ( inoki et al . , 2003b ) . recently , drosophila tctp ( translationally controlled tumour protein ) has been reported as drheb gef ( gtpase exchange factor ) and activates dtor ( hsu et al . , 2007 ) . whether mammalian tctp also can act as rheb gef or not needs to be examined . the first indication that amino acids are involved in mtorc1 pathway was shown in the study of hepatocyte autophagy ( blommaart et al . , 1995 ) in which cellular organelles are encompassed by intracellular double membrane followed by formation of autophagosome - lysosome complex and degraded ( shitani & klionsky , 2004 ) . in the study , addition of amino acids inhibited valine release as a result of autophagic protein degradation in the cell and this inhibition was sharply correlated with the increase of s6 phosphorylation . moreover , then the question is how the level of amino acids is sensed and signals to mtorc1 ( fig . amino acids are imported to the cells via various amino acid transporters on the cell membrane . ( 1992 ; 1994 ) tested the hypothesis whether amino acid signaling is initiated from the membrane transporter using nontransportable peptide leu8-map which contains eight leucine ( the single most effective amino acid ) residues outside and a leucine analogue , isovaleryl - l - carnitine ( ivc ) . treatment of isolated rat hepatocytes with these compounds inhibited macroautophagy and proteolysis , which indicates that leu8-map , ivc , and probably free leucine must be recognized at the plasma membrane or within an associated vacuole . different group , however , reported that only after over 1 hour treatment of the leu8-map could phosphorylate s6 in some degree and moreover , in rat adipocytes , s6k1 and 4ebp1 phosphorylation was unresponsive to leu8-map or ivc ( lynch et al . , 2000 ) . another concern on this hypothesis is that amino acid transporter systems have low specificity or strictness in the kind of amino acids or in structural requirement ( hyde et al . , for example , system l amino acid transporter can import isoleucine , valine , methionine , phenylalanine , and histidine to the cells other than leucine . conversely , pretreatment of 2-amino-3-norbane carboxylic acid ( bch ) , a selective system l amino acid transporter inhibitor , did not inhibit leucine - induced mtorc1 activity because leucine could be imported through other transporter system ( lynch et al . , 2000 ) . on the other hand , it has been suggested that amino acids - induced cell swelling is a mechanism of amino acids - mediated mtorc1 activation . some amino acids are transported into the cell via na+-dependent transporters from which intracellular osmolarity is increased due to imported na+ and accumulated amino acid metabolites ( hyde et al . , 2003 ) . cell swelling itself has shown to exert similar effects as insulin in which protein synthesis is stimulated and protein degradation is inhibited ( reviewed in ( haussinger , 1996 ) ) . moreover , the most effective leucine transporter , system l amino acid transporter , is na+-independent . this suggests that intracellular amino acid concentration or intracellular receptor , but not a particular membrane amino acid transporter , is responsible for the regulation of mtorc1 signaling pathway . bacteria and yeast recognize amino acids starvation by increased concentration of deacylated trna or reduced availability of aminoacylated trna . in jurkat cells , amino acid alcohols which compete with amino acids prevent aminoacyl - trna formation and inhibit s6k1 phosphorylation ( iiboshi et al . , 1999 ) . inhibition of protein synthesis by applying translation inhibitors such as cycloheximide ( chx ) and puromycin has shown to increase s6k1 phosphorylation in the amino acids starvation condition ( iiboshi et al . , 1999 ) . these data suggest that increased level of intracellular amino acids resulted from translation inhibition and of aminoacylated trna is responsible for the amino acid sensing in mammalian cells as well . the level of intracellular amino acids is not changed during short time ( 30 min ) amino acids starvation or stimulation , whereas , s6k1 phosphorylation is changed within this short time frame . moreover , in rat adipocytes , leucine alcohol ( leucinol ) treatment did not affect 4ebp1 phosphorylation ( lynch et al . , 2000 ) . on the other hand suppression of rheb and tor gene expression by rnai in drosophila s2 cells did not affect nutrients ( glucose & bulk amino acids ) import but significantly reduced ribosome biogenesis , protein synthesis , and cell size ( hall et al . , 2007 ) . imported amino acids are subjected for amino acid metabolism and there are several reports suggesting that amino acid metabolites including atp are responsible for mtorc1 activation . for example , leucine is metabolized to -ketoisocaproic acid ( -kic ) by reversible aminotransferase ( at ) , which is then further metabolized to tca cycle intermediates by branched - chain -ketoacid dehydrogenase ( bckdh ) . otherwise , leucine as it is can be an allosteric activator of glutamate metabolizing enzyme , glutamate dehydrogenase ( gdh ) ( fahien et al . , 1988 ; sener & malaisse , 1980 ) . the consequence of leucine metabolic pathways would be production of atp and other metabolically - linked secondary signals , which was proposed as a mechanism of mtor signaling pathway activation by leucine in pancreatic -cells ( mcdaniel et al . , 2002 ) . 2001 ) have previously reported that mtor is an intracellular atp sensor and atp depletion caused by incubation of cells with 2-deoxyglucose or several mitochondria inhibitors including rotenone and antimycin a inhibits s6k1 and 4ebp1 phosphorylation ( inoki et al . a concern on this hypothesis is that the concentration of atp is not affected by short time amino acids deprivation or stimulation in most mammalian cells ( dennis et al . , 2001 ) . nevertheless , it has been shown that an at inhibitor , aminooxyacetic acid ( aoaa ) , inhibits s6k1 phosphorylation upon leucine treatment in pancreatic -cells ( xu et al . , 2001 ) . a nonmetabolized leucine analog ,  (  ) 2-aminobicyclo [ 2.2.1 ] heptane-2-carboxylic acid , also failed to stimulate s6k1 phosphorylation in the absence of leucine ( mcdaniel et al . , 2002 moreover , proteolytic dose responses and inhibition of autophagy to l - leucine and to ivc , readily metabolizable leucine analogue , were similar in the perfused rat liver ( miotto et al . , 1992 ) . stimulation with -kic phosphorylates 4ebp1 in the absence of leucine in adipocytes ( lynch et al . , 2000 ) . taken together , this suggests that although it is difficult to pinpoint exactly what it is , certain products of leucine oxidation are responsible for mtorc1 activation . in the liver , therefore , we still can not exclude a possibility that the effect of leucine on the mtorc1 activation is due to leucine itself at some undefined locus of recognition . leucine metabolizing enzyme , at , is a reversible aminotransferase which also can convert -kic to leucine . interestingly , -kic induced phosphorylation of 4ebp1 was less than leucine induced 4ebp1 phosphorylation and moreover , -kic effect was attenuated in the presence of transaminase inhibitor , l - cycloserine ( lynch et al . , 2000 ) . ( 2000 ) also found that norleucine which is not incorporated in protein translation was as effective as leucine in 4ebp1 phosphorylation , which suggests that there may be a specific intracellular leucine receptor , which recognizes the specific structure of leucine . however , leucine analogues which contain either strong leucine agonist or antagonist activity in one cell line do not always exert the same effect in different cell lines . although the mechanism is not known , it is obvious that amino acids regulate mtorc1 activity ( hara et al . , 1998 ) . that is , amino acids stimulation increases s6k1 and 4ebp1 phosphorylation , whereas , amino acids deprivation rapidly dephosphorylates s6k1 and 4ebp1 . therefore , extensive research efforts have centered on mtorc1 regulation itself . ectopic overexpression of rheb completely inhibited s6k1 and 4ebp1 dephosphorylation upon amino acids withdrawal ( long et al . since rheb activates mtorc1 activity by directly binding to mtor , rheb - mtor interaction was examined . ( 2005b ) showed that rheb - mtor interaction was in fact reduced upon amino acids withdrawal , whereas the interaction was fully recovered when amino acids alone were added to d - pbs . amino acids , however , did not change rheb - raptor and rheb - mlst8 interaction ( long et al . curiously , however , rheb - mtor interaction is not affected by nucleotide binding status of rheb ( long et al . , 2005a ) . the binding of nucleotide - free mutant of rheb to mtor was even stronger than that of wild - type rheb , although it impaired mtor kinase activity . for example , gtp level of overexpressing rheb was increased by amino acids stimulation ( smith et al . , 2005 ) , but endogenous rheb - gtp charging was not changed by amino acids withdrawal ( nobukuni et al . , 2005 ) . ( 2006 ) reported that amino acids starvation did not change rheb - gtp level , but add back amino acids clearly increased rheb - gtp loading . in consistent with this , tsc1/2 is also not required for amino acids - mediated mtorc1 regulation ( roccio et al . , 2006 ; smith et al . , 2005 ) . amino acids withdrawal still dephosphorylated s6k1 and 4ebp1 independent of tsc2 gene expression ( nobukuni et al . , 2005 ; smith et al . , on the other hand , it has been proposed that amino acids regulate mtorc1 activity by modulating mtor - raptor interaction . amino acids starvation causes tighter binding of raptor to mtor which inhibits mtor kinase activity whereas amino acids stimulation loosens the interaction ( kim et al . , 2002 ) . this suggests that amino acids specifically modulate mtor - raptor interaction and regulate mtor kinase activity . recently , several intracellular proteins have been identified as intracellular amino acid sensors which include hvps34 , map4k3 , and rag . vps34 : vps34 is a class iii pi3k which forms an active complex with another kinase vps15 and generates ptdins(3)p by phosphorylating the 3 ' hydroxyl position of the phosphatidylinositol ring ( reviewed in ( backer , 2008 ; nobukuni et al . , 2007 ; vps34 was initially known to play an important role in vesicular trafficking ( lindmo & stenmark , 2006 ; odorizzi et al . , 2000 ) and a role of vps34 in amino acids sensing has been recently reported ( byfield et al . , 2005 ; nobukuni et al . , 2005 ) . that is , hvps34 activity as well as ptdins(3)p production was increased by amino acids stimulation . on the contrary , suppression of hvps34 gene expression with hvps34 sirna or overexpression of gfp - fyve ( fab1p , yotb , vac1p , eea1 ( early endosomal antigen 1 ) ) expression vector which acts as a dominant negative by sequestering ptdins(3)p impaired s6k1 and 4ebp1 phosphorylation but did not affect insulin - stimulated akt phosphorylation . instead , amino acids stimulation facilitates ca influx , which in turn increases the interaction of ca2/cam with the hvps34-mtor and activates mtorc1 ( gulati et al . , 2008 ) . there are several concerns , however , on presenting hvps34 as an amino acids sensing system to activate mtorc1 . ectopic expression of hvps34 to mammalian cells increased s6k phosphorylation only in the presence , but not in the absence of amino acids ( nobukuni et al . , 2005 ) . in the study of yan et al . most of all , in a recent drosophila in vivo study , loss - of - function mutant animals of drosophila vps34 showed disrupted autophagy and endocytosis but tor signaling was not affected by disruption of vps34 gene ( juhasz et al . , 2008 ) . that is , cell size was similar between vps34 mutant and wild - type cells and moreover , modulation of cell size by overexpressing rheb ( increased cell size ) or tsc1/2 ( decreased cell size ) was not affected by vps34 mutation . nevertheless , amino acids starvation induced autophagy in control animals but not in vps34 mutant animals . considering that reported function of hvps34 in amino acid signaling has been all performed in cell culture system , this observation in vivo raises a significant question on the proposed model . ( 2007 ) identified map4k3 protein kinase as an important intracellular protein in amino acids sensing . they showed map4k3 kinase activity was regulated by amino acids and overexpression of map4k3 specifically increases s6k phosphorylation even in the amino acids starvation condition in a tsc1/2 independent manner . this is a difference between hvps34 and map4k3 in amino acids sensing since ectopic expression of hvps34 increases s6k1 phosphorylation only in the presence of amino acids . nevertheless , it would be interesting to address a question whether overexpression of map4k3 increases ptdins(3)p either in the presence or absence of amino acids . rag : most recently , two independent research groups newly discovered that rag ( ras - related gtpases ) small gtpases ( raga , b , c , and d ) play an important role in amino acid signaling ( kim et al . , 2008 ; kim & guan , 2009 ; sancak et al . , s2 cells , knock - down of drag gene expression impaired ds6k phosphorylation upon amino acids stimulation ( kim et al . , 2008 ) . in mammalian cells , expression of gtp - bound active raga / ragb overcomes s6k1 dephosphorylation in amino acids deprivation condition , whereas gdp - bound inactive raga / b inhibited s6k1 phosphorylation upon amino acids stimulation . moreover , draga expressing cells showed bigger cell size than neighboring wild type cells and inhibited starvation - induced autophagy in drosophila . amino acids increase gtp - level of ragb and ragb - ragc heterodimer increases mtorc1 activity by increasing raptor - mtor interaction . on the other hand , in ragb expressing cells , mtor was already present on the rab7 positive peri - nuclear and vesicular structures even in the amino acids deprivation condition . this suggests that the function of rag , mtor , and hvps34 can be interconnected on rab7 positive endosome membrane and may involve in amino acids transporter trafficking to the cell membrane as yeast rag homologue , gtr does ( gao et al . , 2006 ) . 2009 ) found that l - glutamine uptake by solute carrier family1 ( slc1a5 ) transporter is important for l - leucine / essential amino acids ( eaa ) import into the cells . simultaneous efflux of l - glutamine out of cells through slc7a5/slc3a2 , a bidirectional transporter , facilitates l - leucine / eaa import into the cells from which mtorc1 is activated . in contrast to l - glutamine , l - glutamic acid and -ketoglutarate pretreatment could not activate s6k1 upon eaa treatment , which suggests that l - glutamine effect on s6k1 is not due to increasing cellular energy level . interestingly , previously performed large - scale genetic array analysis identified that protein mutations involved in glutamate / glutamine homeostasis also genetically interact with gtr2 ( dubouloz et al . , 2005 ) . lethal phenotype of these mutants was reversed either by glutamate supplement in the medium or by introduction of gtr2-expression plasmids . moreover , gtr2 mutant suppressors identified from genome - wide screen include genes which are directly or indirectly involved in glutamate / glutamine synthesis / degradation . it has been shown that gse complex consisted of five proteins encoded by gtr1 , gtr2 , ybr077c ( ego3 ) , ykr007w ( ego1 ) , and ltv1 genes plays an important role in recycling of general amino acid permease , gap1p , from the late endosome to the plasma membrane in yeast ( gao et al . , 2006 ) . therefore , it will be interesting to address a question whether rag affect slc1a5 and slc7a5/slc3a2 transporter trafficking and whether imported l - leucine / eaa activates mtorc1 through rag . amino acids are imported to the cells via various amino acid transporters on the cell membrane . ( 1992 ; 1994 ) tested the hypothesis whether amino acid signaling is initiated from the membrane transporter using nontransportable peptide leu8-map which contains eight leucine ( the single most effective amino acid ) residues outside and a leucine analogue , isovaleryl - l - carnitine ( ivc ) . treatment of isolated rat hepatocytes with these compounds inhibited macroautophagy and proteolysis , which indicates that leu8-map , ivc , and probably free leucine must be recognized at the plasma membrane or within an associated vacuole . different group , however , reported that only after over 1 hour treatment of the leu8-map could phosphorylate s6 in some degree and moreover , in rat adipocytes , s6k1 and 4ebp1 phosphorylation was unresponsive to leu8-map or ivc ( lynch et al . , 2000 ) . another concern on this hypothesis is that amino acid transporter systems have low specificity or strictness in the kind of amino acids or in structural requirement ( hyde et al . , 2003 ) . for example , system l amino acid transporter can import isoleucine , valine , methionine , phenylalanine , and histidine to the cells other than leucine . conversely , pretreatment of 2-amino-3-norbane carboxylic acid ( bch ) , a selective system l amino acid transporter inhibitor , did not inhibit leucine - induced mtorc1 activity because leucine could be imported through other transporter system ( lynch et al . , 2000 ) . on the other hand , it has been suggested that amino acids - induced cell swelling is a mechanism of amino acids - mediated mtorc1 activation . some amino acids are transported into the cell via na+-dependent transporters from which intracellular osmolarity is increased due to imported na+ and accumulated amino acid metabolites ( hyde et al . , 2003 ) . cell swelling itself has shown to exert similar effects as insulin in which protein synthesis is stimulated and protein degradation is inhibited ( reviewed in ( haussinger , 1996 ) ) . moreover , the most effective leucine transporter , system l amino acid transporter , is na+-independent . this suggests that intracellular amino acid concentration or intracellular receptor , but not a particular membrane amino acid transporter , is responsible for the regulation of mtorc1 signaling pathway . bacteria and yeast recognize amino acids starvation by increased concentration of deacylated trna or reduced availability of aminoacylated trna . in jurkat cells , amino acid alcohols which compete with amino acids prevent aminoacyl - trna formation and inhibit s6k1 phosphorylation ( iiboshi et al . , 1999 ) . inhibition of protein synthesis by applying translation inhibitors such as cycloheximide ( chx ) and puromycin has shown to increase s6k1 phosphorylation in the amino acids starvation condition ( iiboshi et al . , 1999 ) . these data suggest that increased level of intracellular amino acids resulted from translation inhibition and of aminoacylated trna is responsible for the amino acid sensing in mammalian cells as well . however , this is not consistent with time frame in which mtorc1 is activated . the level of intracellular amino acids is not changed during short time ( 30 min ) amino acids starvation or stimulation , whereas , s6k1 phosphorylation is changed within this short time frame . moreover , in rat adipocytes , leucine alcohol ( leucinol ) treatment did not affect 4ebp1 phosphorylation ( lynch et al . , 2000 ) . on the other hand suppression of rheb and tor gene expression by rnai in drosophila s2 cells did not affect nutrients ( glucose & bulk amino acids ) import but significantly reduced ribosome biogenesis , protein synthesis , and cell size ( hall et al . , 2007 ) . imported amino acids are subjected for amino acid metabolism and there are several reports suggesting that amino acid metabolites including atp are responsible for mtorc1 activation . for example , leucine is metabolized to -ketoisocaproic acid ( -kic ) by reversible aminotransferase ( at ) , which is then further metabolized to tca cycle intermediates by branched - chain -ketoacid dehydrogenase ( bckdh ) . otherwise , leucine as it is can be an allosteric activator of glutamate metabolizing enzyme , glutamate dehydrogenase ( gdh ) ( fahien et al . , 1988 ; sener & malaisse , 1980 ) . the consequence of leucine metabolic pathways would be production of atp and other metabolically - linked secondary signals , which was proposed as a mechanism of mtor signaling pathway activation by leucine in pancreatic -cells ( mcdaniel et al . , 2002 ) . ( 2001 ) have previously reported that mtor is an intracellular atp sensor and atp depletion caused by incubation of cells with 2-deoxyglucose or several mitochondria inhibitors including rotenone and antimycin a inhibits s6k1 and 4ebp1 phosphorylation ( inoki et al . a concern on this hypothesis is that the concentration of atp is not affected by short time amino acids deprivation or stimulation in most mammalian cells ( dennis et al . , 2001 ) . nevertheless , it has been shown that an at inhibitor , aminooxyacetic acid ( aoaa ) , inhibits s6k1 phosphorylation upon leucine treatment in pancreatic -cells ( xu et al . , 2001 ) . a nonmetabolized leucine analog ,  (  ) 2-aminobicyclo [ 2.2.1 ] heptane-2-carboxylic acid , also failed to stimulate s6k1 phosphorylation in the absence of leucine ( mcdaniel et al . , 2002 moreover , proteolytic dose responses and inhibition of autophagy to l - leucine and to ivc , readily metabolizable leucine analogue , were similar in the perfused rat liver ( miotto et al . , 1992 ) . stimulation with -kic phosphorylates 4ebp1 in the absence of leucine in adipocytes ( lynch et al . , 2000 ) . taken together , this suggests that although it is difficult to pinpoint exactly what it is , certain products of leucine oxidation are responsible for mtorc1 activation . in the liver , therefore , we still can not exclude a possibility that the effect of leucine on the mtorc1 activation is due to leucine itself at some undefined locus of recognition . leucine metabolizing enzyme , at , is a reversible aminotransferase which also can convert -kic to leucine . interestingly , -kic induced phosphorylation of 4ebp1 was less than leucine induced 4ebp1 phosphorylation and moreover , -kic effect was attenuated in the presence of transaminase inhibitor , l - cycloserine ( lynch et al . , 2000 ) . ( 2000 ) also found that norleucine which is not incorporated in protein translation was as effective as leucine in 4ebp1 phosphorylation , which suggests that there may be a specific intracellular leucine receptor , which recognizes the specific structure of leucine . however , leucine analogues which contain either strong leucine agonist or antagonist activity in one cell line do not always exert the same effect in different cell lines . although the mechanism is not known , it is obvious that amino acids regulate mtorc1 activity ( hara et al . , 1998 ) . that is , amino acids stimulation increases s6k1 and 4ebp1 phosphorylation , whereas , amino acids deprivation rapidly dephosphorylates s6k1 and 4ebp1 . ectopic overexpression of rheb completely inhibited s6k1 and 4ebp1 dephosphorylation upon amino acids withdrawal ( long et al . since rheb activates mtorc1 activity by directly binding to mtor , rheb - mtor interaction was examined . 2005b ) showed that rheb - mtor interaction was in fact reduced upon amino acids withdrawal , whereas the interaction was fully recovered when amino acids alone were added to d - pbs . amino acids , however , did not change rheb - raptor and rheb - mlst8 interaction ( long et al . , curiously , however , rheb - mtor interaction is not affected by nucleotide binding status of rheb ( long et al . , 2005a ) . the binding of nucleotide - free mutant of rheb to mtor was even stronger than that of wild - type rheb , although it impaired mtor kinase activity . for example , gtp level of overexpressing rheb was increased by amino acids stimulation ( smith et al . , 2005 ) , but endogenous rheb - gtp charging was not changed by amino acids withdrawal ( nobukuni et al . , 2005 ) . ( 2006 ) reported that amino acids starvation did not change rheb - gtp level , but add back amino acids clearly increased rheb - gtp loading . in consistent with this , tsc1/2 is also not required for amino acids - mediated mtorc1 regulation ( roccio et al . amino acids withdrawal still dephosphorylated s6k1 and 4ebp1 independent of tsc2 gene expression ( nobukuni et al . , 2005 ; on the other hand , it has been proposed that amino acids regulate mtorc1 activity by modulating mtor - raptor interaction . amino acids starvation causes tighter binding of raptor to mtor which inhibits mtor kinase activity whereas amino acids stimulation loosens the interaction ( kim et al . , 2002 ) . this suggests that amino acids specifically modulate mtor - raptor interaction and regulate mtor kinase activity . recently , several intracellular proteins have been identified as intracellular amino acid sensors which include hvps34 , map4k3 , and rag . vps34 : vps34 is a class iii pi3k which forms an active complex with another kinase vps15 and generates ptdins(3)p by phosphorylating the 3 ' hydroxyl position of the phosphatidylinositol ring ( reviewed in ( backer , 2008 ; nobukuni et al . , 2007 ; yan & backer , 2007 ) ) . vps34 was initially known to play an important role in vesicular trafficking ( lindmo & stenmark , 2006 ; odorizzi et al . , 2000 ) and a role of vps34 in amino acids sensing has been recently reported ( byfield et al . , 2005 ; nobukuni et al . , 2005 ) . that is , hvps34 activity as well as ptdins(3)p production was increased by amino acids stimulation . on the contrary , suppression of hvps34 gene expression with hvps34 sirna or overexpression of gfp - fyve ( fab1p , yotb , vac1p , eea1 ( early endosomal antigen 1 ) ) expression vector which acts as a dominant negative by sequestering ptdins(3)p impaired s6k1 and 4ebp1 phosphorylation but did not affect insulin - stimulated akt phosphorylation . instead , amino acids stimulation facilitates ca influx , which in turn increases the interaction of ca2/cam with the hvps34-mtor and activates mtorc1 ( gulati et al . , 2008 ) . there are several concerns , however , on presenting hvps34 as an amino acids sensing system to activate mtorc1 . ectopic expression of hvps34 to mammalian cells increased s6k phosphorylation only in the presence , but not in the absence of amino acids ( nobukuni et al . , 2005 ) . most of all , in a recent drosophila in vivo study , loss - of - function mutant animals of drosophila vps34 showed disrupted autophagy and endocytosis but tor signaling was not affected by disruption of vps34 gene ( juhasz et al . , 2008 ) . that is , cell size was similar between vps34 mutant and wild - type cells and moreover , modulation of cell size by overexpressing rheb ( increased cell size ) or tsc1/2 ( decreased cell size ) was not affected by vps34 mutation . nevertheless , amino acids starvation induced autophagy in control animals but not in vps34 mutant animals . considering that reported function of hvps34 in amino acid signaling has been all performed in cell culture system , this observation in vivo raises a significant question on the proposed model . ( 2007 ) identified map4k3 protein kinase as an important intracellular protein in amino acids sensing . they showed map4k3 kinase activity was regulated by amino acids and overexpression of map4k3 specifically increases s6k phosphorylation even in the amino acids starvation condition in a tsc1/2 independent manner . this is a difference between hvps34 and map4k3 in amino acids sensing since ectopic expression of hvps34 increases s6k1 phosphorylation only in the presence of amino acids . nevertheless , it would be interesting to address a question whether overexpression of map4k3 increases ptdins(3)p either in the presence or absence of amino acids . rag : most recently , two independent research groups newly discovered that rag ( ras - related gtpases ) small gtpases ( raga , b , c , and d ) play an important role in amino acid signaling ( kim et al . , 2008 ; kim & guan , 2009 ; sancak et al . , s2 cells , knock - down of drag gene expression impaired ds6k phosphorylation upon amino acids stimulation ( kim et al . , expression of gtp - bound active raga / ragb overcomes s6k1 dephosphorylation in amino acids deprivation condition , whereas gdp - bound inactive raga / b inhibited s6k1 phosphorylation upon amino acids stimulation . moreover , draga expressing cells showed bigger cell size than neighboring wild type cells and inhibited starvation - induced autophagy in drosophila . amino acids increase gtp - level of ragb and ragb - ragc heterodimer increases mtorc1 activity by increasing raptor - mtor interaction . on the other hand , in ragb expressing cells , mtor was already present on the rab7 positive peri - nuclear and vesicular structures even in the amino acids deprivation condition . this suggests that the function of rag , mtor , and hvps34 can be interconnected on rab7 positive endosome membrane and may involve in amino acids transporter trafficking to the cell membrane as yeast rag homologue , gtr does ( gao et al . , 2006 ) . 2009 ) found that l - glutamine uptake by solute carrier family1 ( slc1a5 ) transporter is important for l - leucine / essential amino acids ( eaa ) import into the cells . simultaneous efflux of l - glutamine out of cells through slc7a5/slc3a2 , a bidirectional transporter , facilitates l - leucine / eaa import into the cells from which mtorc1 is activated . in contrast to l - glutamine , l - glutamic acid and -ketoglutarate pretreatment could not activate s6k1 upon eaa treatment , which suggests that l - glutamine effect on s6k1 is not due to increasing cellular energy level . interestingly , previously performed large - scale genetic array analysis identified that protein mutations involved in glutamate / glutamine homeostasis also genetically interact with gtr2 ( dubouloz et al . , 2005 ) . lethal phenotype of these mutants was reversed either by glutamate supplement in the medium or by introduction of gtr2-expression plasmids . moreover , gtr2 mutant suppressors identified from genome - wide screen include genes which are directly or indirectly involved in glutamate / glutamine synthesis / degradation . it has been shown that gse complex consisted of five proteins encoded by gtr1 , gtr2 , ybr077c ( ego3 ) , ykr007w ( ego1 ) , and ltv1 genes plays an important role in recycling of general amino acid permease , gap1p , from the late endosome to the plasma membrane in yeast ( gao et al . , 2006 ) . therefore , it will be interesting to address a question whether rag affect slc1a5 and slc7a5/slc3a2 transporter trafficking and whether imported l - leucine / eaa activates mtorc1 through rag . how amino acids regulate protein synthesis and cell growth has been a fundamental biological question in the field . since inhibition of tor pathway mimics amino acids starvation condition and moreover , amino acids are the most potent activator of tor , extensive research efforts have centered on elucidating a mechanism of amino acids sensing in mtor signaling pathway . from the cell membrane to intracellular proteins , each path of amino acids has been examined for a possible amino acid sensing point , however , there have been still many controversies in experimental data due to such a structural and metabolic diversity of amino acids . recent identification of hvps34 , map4k3 , and rag gtpases as important intracellular proteins in amino acid signaling and of a mechanism of l - leucine / eaa import into the cells made a significant progress in our understanding of amino acids - mediated mtor activation . however , how imported amino acids are sensed by hvps34 , map4k3 , or rag gtpases and activate mtorc1 still remains as an open question . the importance of understanding amino acid signaling is that it has many significant physiological and pathological implications in vivo . anabolic response of the body after protein meal ingestion is closely correlated with phosphorylation of s6k1 and 4ebp1 in mice , rat , and human ( balage et al . , 2001 ; long et al . , 2000 insulin production in pancreatic -cells is increased by amino acids stimulation ( may & buse , 1989 ) and amino acids infusion increases the sensitivity of muscle protein synthesis to insulin ( garlick & grant , 1988 ) . furthermore , activated s6k in turn phosphorylates and degrades insulin receptor substrate ( irs ) and inhibits pi3k signaling pathway as a negative feedback inhibition , which can decrease insulin sensitivity and lead to diabetes ( fig . 1 ) . overactivated mtorc1 signaling pathway is frequently found in various cancers and tissue / organ hypertrophy as well ( guertin & sabatini , 2007 ; inoki et al . , 2005b ; lee et al . , 2007 although rapamycin has been used as an fda - approved drug to treat some of the cancers including sarcoma , suppression of s6k phosphorylation can result in activating pi3k signaling which can further exacerbate the disease state . therefore , if we elucidate a mechanism on the amino acid signaling pathway to mtorc1 activation , more specific and effective way to prevent or treat these diseases can be developed .
amino acids are fundamental nutrients for protein synthesis and cell growth ( increase in cell size ) . 
 recently , many compelling evidences have shown that the level of amino acids is sensed by extra- or intra - cellular amino acids sensor(s ) and regulates protein synthesis / degradation . 
 mammalian target of rapamycin complex 1 ( mtorc1 ) is placed in a central position in cell growth regulation and dysregulation of mtor signaling pathway has been implicated in many serious human diseases including cancer , diabetes , and tissue hypertrophy . 
 although amino acids are the most potent activator of mtorc1 , how amino acids activate mtor signaling pathway is still largely unknown . 
 this is partly because of the diversity of amino acids themselves including structure and metabolism . in this review , 
 current proposed amino acid sensing mechanisms to regulate mtorc1 and the evidences pro / against the proposed models are discussed .