Indice

Indice
Aim
Introduction
Involved proteins
Phosphate role
Sugar Role
Insulin stimolation
Amino acids role
mTOR
What does mTOR induce?
Conclusions
Bibliografia

Aim
What we are wondering is if  hamartin-tuberin proteic complex and mTOR have a role in cellular cycle regulation and if it is able to settle for any molecule.
We know that presence of this proteic complex in the cell forbids ciclyne signal and cell cycle progression through mTOR. 
Since the cell proceeds to mitosis it is necessary that cells have everything them need. Those molecules could be: amino acids, sugar, phosphate and oxygen. We know that sugar is settle by insulin and phosphate is settled by  ATP. So we presume that the amount of the complex in cells sets for amino acids level. 
Moreover we can wonder if also eme and Fe plays any kind of role. Actually TSC1-TSC2 product consistently with HIF regulates also VEGF production. HIF in order to stimulate VEGF need eme and hormones. The fact that the hamartin-tuberin complex is in the cell could mean a lack Fe and eme or the presence of oxygen. [1]

Introduction
Involved proteins

TSC1 on chromosome 9q34 is the gene that encodes for hamartin, a protein with a molecular mass of 130,000 (130K) that contains coiled-coil domains, but no obvious catalytic domains. TSC2 on chromosome 16p13 is the gene that encodes for tuberin, a 200K protein that contains a coiled-coil domain and a carboxy-terminal region, which shares homology to the Rap GTPase-activating protein (GAP). 
Tuberin and hamartin interact and appear to function as a complex to inhibit signalling by the mammalian target of rapamycin (mTOR).
In mice, homozygous inactivation of either TSC1 or TSC2 is embryonic lethal, whereas heterozygous animals are prone to tumours. 
Mutations in two genes, TSC1 and TSC2, cause Tuberous sclerosis, a desease in which cells loose proliferation control. [2]

Phosphate role
TSC1- Hamartin
Endogenous hamartin is threonine-phosphorylated during the G2/M phase of a normal cell cycle, so hamartin, like tuberin, is regulated by phosphorylation. Results from both in vitro and in vivo experiments indicate that hamartin is a substrate of the cyclin-dependent kinase CDK1 (cdc2), which is active in late cell cycle phases and promotes entry into mitosis when bound to cyclin B1. Hamartin is phosphorylated by CDK1 at three residues, the most highly conserved of which lies within the hamartin-tuberin interaction domain (Thr417 -hamartin-tuberin interaction domain-, Ser584, and Thr1047). A mutant form of hamartin that cannot be phosphorylated by CDK1 increased the inhibition of p70S6K by the hamartin-tuberin complex. This suggests that regulation of the hamartin-tuberin complex during G2/M may play a role in the integration of cell division with cell size, protein synthesis, and/or growth factor signaling.
Both in vitro and in vivo studies indicate that hamartin is a direct target for phosphorylation by CDK1. In vivo, hamartin phosphorylation is blocked by the CDK1 inhibitor alsterpaullone, and hamartin with the three residues mutated to alanine (TSC1AAA) is not threonine-proline-phosphorylated during nocodazole-induced G2/M arrest or during G2/M progression of the normal cell cycle. 
The fact that endogenous hamartin is phosphorylated during the normal cell cycle suggests that hamartin phosphorylation has physiologic significance. 
It is possible that phosphorylation of hamartin by CDK1 in the G2/M phase regulates the activity, rather than the interaction, of the hamartin-tuberin complex during the G2/M phase and mitosis, so the phosphorylation of hamartin by CDK1 could play a central role in the coordination of cell cycle progression, cell growth, and cell size. [2] [3] [12] (Fig. 1).

Fig. 1. Schematic representation of the structural-functional domains of hamartin.
Hamartin has potential transmembrane and coiled-coil domains; among functional domains are the tuberin-binding, ERM-binding, neurofilament-L-binding, and Rho-activating domains. [7]

TSC2- Tuberin
Tuberin has a highly conserved domain with homology to Rap1 GTPase activating protein, and it has been shown to possess GTPase activating protein activity for Rap1, Rab5, and Rheb. 
Tuberin is phosphorylated by the kinase Akt (protein kinase B). Phosphorylation of tuberin by Akt regulates 2 protein: 40S ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) by tuberin. Tuberin is also a substrate of the p38 and MK2 kinase cascade.
The GTPase-activating protein (GAP) domain found in the C terminus of TSC2 is the only functional domain identified in TSC1/2. GAPs down regulate the activity of small G proteins (small GTP-binding proteins). Small G proteins are molecular switches that are in an active state when bound to GTP and become inactivate by hydrolysis of GTP to GDP. In their GTP-bound active state, small G proteins can interact with a variety of effector proteins. By directly or indirectly altering the function of these effectors, small G-proteins serve as molecular ‘gatekeepers’ to regulate various biological functions. The cycling between active GTP- and inactive GDP-bound states is controlled by guanine nucleotide exchange factors (GEFs), which activate small G proteins by promoting exchange of GDP for GTP, and also by GAPs, which cause inactivation of small G proteins by enhancing their low intrinsic GTPase activity. 
TSC2 increases the intrinsic GTPase activity of both Rap1 and Rab5 in vitro. But it is not known whether this activity occurs in vivo or if TSC2 activity on Rap1 or Rab5 contributes to the physiological function of the TSC1/2 complex. [3] (Fig. 2).

Fig. 2. Schematic representation of the structural-functional domains of tuberin.
Tuberin includes leucine zipper, two coiled-coil, two transcription-activating domains (TAD), GAP homology, and calmodulin (CaM)-binding domains. Hamartin-binding motive is localized at the N-terminal; multiple Akt/PKB-dependent phosphorylation sites are indicated with arrows; phosphorylation sites modulating 14-3-3 association are also indicated with arrows. [7]
Sugar Role
Insulin stimolation
Engagement of insulin receptor by insulin normally leads to recruitment of the IRS-1 protein, which subsequently recruits PI3K. 
Active PI3K subsequently converts phosphatidylinositol-4,5-phosphate (PIP2) in the cell membrane to phosphatidylinositol-3,4,5-phosphate (PIP3), which binds kinases containing a plextrin-homology domain such as phosphoinositide-dependent kinase 1 (PDK1) and Akt, and recruits them to the membrane. PDK1 both phosphorylates S6K1 directly and activates Akt, which signals to 4EBP1 (eIF4E binding protein 1; eIF4E, eukaryotic initiation factor 4E) and S6K1 (and the S6 ribosomal protein kinase 1) through a mechanism not completely understood.
Starvation of cells for amino acids, glucose, or ATP (by inhibition of glycolysis), and the induction of mitochondrial dysfunction by various chemical agents, have both been reported to inhibit the growth factor-induced activation of 4EBP1 and S6K1. Integrating such a nutrient-sensing mechanism into the molecular model of 4EBP1 and S6K1 regulation by growth factors relied on work examining the small of mTOR (mammalian target of rapamicin). These data give rise to two possible models for how TOR functions in the network. A simple and prevalent model accounting for these observations places TOR downstream of PI3K/Akt in a linear fashion. This model is consistent with the existence of an insulin-sensitive phosphorylation site in TOR. However, an alternative model places insulin and TOR in parallel pathways, both of which are required to activate S6K1. Several detailed studies of S6K1 regulation are consistent with the latter model. 
TOR may directly phosphorylate S6K1 and may even signal to S6K1 through a combination of the above two mechanism.
The fact that nutrients modulate a phosphorylation site within TOR (Ser2448) argues that TOR itself receives signals from nutrients, consistent with a model in which it relays the nutrient signal to effectors such as 4EBP1/S6K1. Thus, a model emerged in which cell growth effectors such as 4EBP1 and S6K1 lie at the intersection of two regulatory signals: a growth factor signal carried by PI3K (and potentially in part by TOR) and a nutrient signal (amino acids, glucose) carried by TOR. Although 4EBP1 and S6K1 have been well characterized, it is possible that the PI3K/TOR network regulates additional effectors to control cell growth. [4] (Fig. 3-4).

Figure 3. Generic PI 3-kinase–Akt and nutrient–TOR signaling pathways.
(A) In the absence of extracellular growth signals (‘OFF’), translation rates are low because TSC1–TSC2 inhibits TOR and 4E-BP1 inhibits
eIF4E. (B) In the presence of growth factors (‘ON’), PI 3-kinase–Akt signaling is activated. Phosphorylation of TSC2 by Akt promotes its
binding to 14-3-3 proteins, somehow inactivating the TSC1–TSC2 complex. This derepresses TOR, leading to phosphorylation of 4EBP1
and S6 kinase and thus increased translation. TOR activity is also promoted by nutritional inputs, though how is not clear. Note that this is an oversimplified model and several components have additional upstream inputs and downstream targets not shown. 
Abbreviations: In, insulin; IGF, insulin-like growth factors; InR, insulin/IGF receptor; IRS, insulin receptor substrate; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PDK1, phosphoinositide-dependent kinase 1; 4E-BP1, eIF4E binding protein 1; eIF4E, eukaryotic initiation factor 4E; 5′TOP, 5′ terminal oligopyrimidine tract-containing mRNA. Green arrows indicate activation and red arrows indicate inhibition. Colored or grayed shapes represent active or inactive molecules, respectively. Circled ‘P’s represent phosphorylation events. [9]

The mechanism by which phosphorylation of TSC2 by Akt leads to activation of S6K1 is unclear. Two groups have shown that phosphorylation of TSC2 disrupts its association with TSC1. This event could cancel the repressive effect of hamartin-tuberin proteic complex. (Fig. 4)

Fig. 4. Illustration of negative regulation of TSC tumor suppressor complex by PI3K/Akt. [14]
Akt regulates growth by phosphorylating and inhibiting the Tsc1–Tsc2 complex. Akt activity, either by activation of IR, PI(3)K or loss of PTEN, results in direct phosphorylation of Tsc2. This event disrupts binding to Tsc1 and thereby inactivates the Tsc complex. Inactivation of the Tsc1–Tsc2 complex results in increased cell growth, possibly mediated by downstream inhibition of TOR and activation of S6K18. [16]

Inactivity of mTOR could lead to growth arrest in the the G1 phase of the cell so blockage of mTOR can inhibit cell cycle traverse at the G1/S phase transition [15]. (Fig 5).

Fig. 5. Mechanism of tuberin and hamartin cell signalling.
 Tuberin and hamartin form a cytosolic heteromer, which may also contain a yet to be identified protein(s); growth factor-mediated stimulation of PI 3-kinase followed by Akt/PKB activation leads to phosphorylation of tuberin, which, in turn, releasesS6K1 from negative regulation. In parallel, the tuberin and hamartin complex negatively regulates nutrient-mediated activation of mTOR and phosphorylation of 4E-BP1. [7].
Amino acids role 
mTOR

mTOR is a protein encoded by FRAP1 gene on chromosome 1. It is a kinase involved in cell cycle progression. It is a sensor of aminoacidic level. Here we report his amino acid composition.

Aminoacid sequence of mTOR

GAGGLKRRYRCWRRQLRPWPKPREPQGKMLGTGPAAATTAATTSSNVSVLQQFASGLKSRNEETRAKAAKELQHYVTMELREMSQEESTRFYDQLNHHIFELVSSSDANERKGGILAIASLIGVEGGNATRIGRFANYLRNLLPSNDPVVMEMASKAIGRLAMAGDTFTAEYVEFEVKRALEWLGADRNEGRRHAAVLVLRELAISVPTFFFQQVQPFFDNIFVAVWDPKQAIREGAVAALRACLILTTQREPKEMQKPQWYRHTFEEAEKGFDETLAKEKGMNRDDRIHGALLILNELV
RISSMEGERLREEMEEITQQQLVHDKYCKDLMGFGTKPRHITPFTSFQAVQPQQSNALVGLLGYSSHQGLMGFGTSPSPAKSTLVESRCCRDLMEEKFDQVCQWVLKCRNSKNSLIQMTILNLLPRLAAFRPSAFTDTQYLQDTMNHVLSCVKKEKERTAAFQALGLLSVAVRSEFKVYLPRVLDIIRAALPPKDFAHKRQKAMQVDATVFTCISMLARAMGPGIQQDIKELLEPMLAVGLSPALTAVLYDLSRQIPQLKKDIQDGLLKMLSLVLMHKPLRHPGMPKGLAHQLASPGLTT
LPEASDVGSITLALRTLGSFEFEGHSLTQFVRHCADHFLNSEHKEIRMEAARTCSRLLTPSIHLISGHAHVVSQTAVQVVADVLSKLLVVGITDPDPDIRYCVLASLDERFDAHLAQAENLQALFVALNDQVFEIRELAICTVGRLSSMNPAFVMPFLRKMLIQILTELEHSGIGRIKEQSARMLGHLVSNAPRLIRPYMEPILKALILKLKDPDPDPNPGVINNVLATIGELAQVSGLEMRKWVDELFIIIMDMLQDSSLLAKRQVALWTLGQLVASTGYVVEPYRKYPTLLEVLLNFLKTEQNQGTRREAIRVLGLLGALDPYKHKVNIGMIDQSRDASAVSLSESKSSQDSSDYSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFIFKSLGLKCVQFLPQVMPTFLNVIRVCDGAIREFLFQQLGMLVSFVKSHIRPYMDEIVTLMREFWVMNTSIQSTIILLIEQIVVALGGEFKLYLPQLIPHMLRVFMHDNSPGRIVSIKLLAAIQLFGANLDDYLHLLLPPIVKLFDAPEAPLPSRKAALETVDRLTESLDFTDYASRIIHPIVRTLDQSPELRSTAMDTLSSLVFQLGKKYQIFIPMVNKVLVRHRINHQRYDVLICRIVKGYTLADEEEDPLIYQHRMLRSGQGDALASGPVETGPMKKLHVSTINLQKATAWGAARRVSKDDWLEWLRRLSLELLKDSSSPSLRSCWALAQAYNPMARDLFNAAFVSCWSELNEDQQDELIRSIELALTSQDIAEVTQTLLNLAEFMEHSDKGPLPLRDDNGIVLLGERAAKCRAYAKALHYKELEFQKGPTPAILESLISINNKLQQPEAAAGVLEYAMKHFGELEIQATWYEKLHEWEDALVAYDKKMDTNKDDPELMLGRMRCLEALGEWGQLHQQCCEKWTLVNDETQAKMARMAAAAAWGLGQWDSMEEYTCMIPRDTHDGAFYRAVLALHQDLFSLAQQCIDKARDLLDAELTAMAGESYSRAYGAMVSCHMLSELEEVIQYKLVPERREIIRQIWWERLQGCQRIVEDWQKILMVRSLVVSPHEDMRTWLKYASLCGKSGRLALAHKTLVLLLGVDPSRQLDHPLPTVHPQVTYAYMKNMWKSARKIDAFQHMQHFVQTMQQQAQHAIATEDQQHKQELHKLMARCFLKLGEWQLNLQGINESTIPKVLQYYSAATEHDRSWYKAWHAWAVMNFEAVLHYKHQNQARDEKKKLRHASGANITNATTAATTAATATTTASTEGSNSESEAESTENSPTPSPLQKKVTEDLSKTLLMYTVPAVQGFFRSISLSRGNNLQDTLRVLTLWFDYGHWPDVNEALVEGVKAIQIDTWLQVIPQLIARIDTPRPLVGRLIHQLLTDIGRYHPQALIYPLTVASKSTTTARHNAANKILKNMCEHSNTLVQQAMMVSEELIRVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQLPQLTSLELQYVSPKLLMCRDLELAVPGTYDPNQPIIRIQSIAPSLQVITSKQRPRKLTLMGSNGHEFVFLLKGHEDLRQDERVMQLFGLVNTLLANDPTSLRKNLSIQRYAVIPLSTNSGLIGWVPHCDTLHALIRDYREKKKILLNIEHRIMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAKLLWLKSPSSEVWFDRRTNYTRSLAVMSMVGYILGLGDRHPSNLMLDRLSGKILHIDFGDCFEVAMTREKFPEKIPFRLTRMLTNAMEVTGLDGNYRITCHTVMEVLREHKDSVMAVLEAFVYDPLLNWRLMDTNTKGNKRSRTRTDSYSAGQSVEILDGVELGEPAHKKTGTTVPESIHSFIGDGLVKPEALNKKAIQIINRVRDKLTGRDFSHDDTLDVPTQVELLIKQATSHENLCQCYIGWCPFW
[5]

As we can see the protein has a intermediate level of triptophan, so this is not the most important component that mTOR sets for. So we had a look to another important amino acid which is methionine and we found that mTOR has high level of  it. Presence of mTOR in the cell means that it has enough methionine to duplicate, but could be a lack of other amino acidis. [17] [18]. (Fig. 6).

Figure 6. Tuberous sclerosis complex 1/2 heterodimer protein (TSC1/2) and Rheb (Ras homolog enriched in brain) are upstream components of the mTOR (mammalian target of rapamycin) signaling pathway. 
Tuberous sclerosis complex 2 (TSC2) is phosphorylated and inhibited by the pro-growth protein AKT, which is activated by growth factors such as insulin. TSC2 is a GTPase-activating protein (GAP) for the small G protein Rheb. Inactivation of Rheb by TSC2 is significant because GTP-bound, activated Rheb leads to an increase in the activation of mTOR, a central regulator of cell growth. In response to amino acids and cellular energy levels, mTOR controls translation through activation of S6K1 (p70 ribosomal protein S6 kinase) and inhibition of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). It is known that cellular nutrient levels (intracellular amino acids) can regulate the function of mTOR, but it remains to be seen what roles TSC1/2 and Rheb have in the intracellular amino acid and energy-sensing network. [3]

TOR could sense nutrients independently of TSC1/2. The mechanism by which TSC1/2 controls TOR function remains unclear. Overexpressed TSC1/2 associate with TOR; Rheb may form part of this complex and affect TOR function either directly or via an associated factor, such as raptor. Another possibility that has been suggested is that Rheb may stimulate TOR by increasing the levels of amino acids within the cell itself. Thus, while the precise mechanism remains controversial, a reasonable model is that phosphorylation of TSC2 by Akt relieves inhibition of TOR by the TSC complex, thereby allowing phosphorylation of 4EBP1 and S6K1. 
The phosphorylation state of TSC2 is nutrient sensitive and the phosphorylation state of TSC2 determines the nutrient signal to S6K1. Because starvation does not modulate the activity of Akt, the nutrient signal would have to affect TSC2 phosphorylation through a novel pathway, perhaps by blocking its ability to act as a substrate for Akt or by modulating the activity of a TSC2 phosphatase. It is also possible that amino acid starvation causes posttranslational modification of the TSC complex at a not yet characterized site. (Fig. 7).

Fig. 7. Possible pathway of regulation throw amino acids. 
Figure on the left. The figure depicts signalling pathways involved in the activation of mRNA translation by stimuli acting through cell surface receptors. These pathways bring about the stimulation of peptide chain initiation or elongation by activating the translation factors or other proteins involved in these processes, by altering their states of phosphorylation. The two protein kinases shown in red (GSK3 and eEF2 kinase) are inactivated as part of this process. Amino acids positively regulate the signalling events linked to the mammalian target of rapamycin, mTOR. eIF4E and eIF2B are eukaryotic initiation factors; S6 is a protein of the small ribosomal subunit; eEF2 is elongation factor 2. [10].
Figure on the right. Three possible models of TSC1–TSC2 function in amino-acid–TOR signaling, based on several studies in Drosophila and mammalian cells. TSC1–TSC2 could either function as obligatory components between amino acids and TOR in a linear amino-acid-sensing pathway (a), or negatively regulate the ability of TOR to sense amino acid levels (b). (c) In third model, TSC1–TSC2 complex act in parallel with TOR to control S6K and 4E-BP. This model postulates that TSC1–TSC2 signals through an unknown protein (X) that, similar to TOR, also regulates both S6K and 4E-BP. 4E-BP, initiation factor 4E-binding protein; S6K, ribosomal subunit S6 kinase (S6K); TOR, target of rapamycin; TSC, tuberous sclerosis complex.[13].

This figure can summarize the role of hamartin-tuberin complex and mTOR. (Fig. 8).

Figure 8. A Model for the Dual Regulation of 4EBP1/S6K1 by Growth Factors and Nutrients. 
Growth factors signal to 4EBP1/S6K1 via PI3K. Nutrients signal to 4EBP1/S6K1 via TOR, independently from PI3K. The PTEN tumor suppressor restrains growth factor signals to 4EBP1/S6K1, and loss of PTEN function leads to hyperactivation of S6K1 and tumor formation. The TSC1/2 tumor suppressors restrain nutrient signals to 4EBP1/S6K1, and loss of TSC1/2 function leads to hyperphosphorylation of 4EBP1/S6K1 and tumor formation. The TSC proteins are themselves regulated by growth factor signals, constituting a point of crosstalk between the growth factor-regulated and nutrient-regulated signaling. A curious feature of the network is the predominance of activating steps on the growth factor pathway (symbolized by the arrows) and inhibiting steps on the nutrient pathway (symbolized by the T bars), perhaps reflecting a braking mechanism to permit growth factor signaling under nutrient-rich conditions but to dominantly override growth factor signaling under nutrient-poor conditions. [4]
What does mTOR induce?

TSC2, by virtue of its ability to inhibit mTOR, down regulates the HIF transcription factor. 
HIF (hypoxia-inducible factor) is a transcriptional complex that mediates a broad range of cellular and systemic responses to hypoxia. In response to hypoxia, stimulation of growth factors, and activation of oncogenes as well as carcinogens, HIF-1alpha is over expressed and/or activated and targets those genes which are required for angiogenesis, metabolic adaptation to low oxygen and promotes survival.
HIF activates a variety of genes implicated in tumorigenesis, including VEGF (vascular endothelial growth factor). Furthermore due to the fact that is also stimulated the production of VEGF  with is able to enhance blood support and disregarding that cells already has amino acids, sugar and phosphate, it could be that the cell still need more oxygen for survival.
So we can presume that hamartin-tuberin complex is also involved in the detection of iron level. [8] (Fig. 9). 

Fig. 9. The fate of HIF at different oxygen levels. Role of HIF in the cell.  [11]

Conclusions 

When mTOR is in high level in the cell we know that there is enough methionine, but it I possible that the cell still need some nutrients or others amino acids. 
When growth signal stimulate the cell mTOR is phosphorilated and it is able to transduct a signal that promote VEGF transcription. VEGF is able to increase blood support and  carry in the cell other proteins which can be catalysed to obtain lacking amino acids. 
In absence of met TNF (which has low level of met) is able to inhibit cell growth because disrupt insulin signal to gain time and allow catabolism of muscle cells to obtain met. 
Presence of methionine allow transduction of insulin signal, mTOR phosphorilation and so VEGF production. (Fig. 10). [17] [18].

Fig. 10. mTOR  role.
mTOR has an important role in sensing intracellular level of methionine and allow cell cycle progression in order to obtain other amino acids. TNF-α is able to block cell cycle progression in order to gain the necessary amount of methionine from the catalysis of musclolar proteins.

Bibliografia

1.	TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling Ken Inoki, Yong Li, Tianquan Zhu, Jun Wu and Kun-Liang Guan
2.	Cell Cycle-regulated Phosphorylation of Hamartin, the Product of the Tuberous Sclerosis Complex 1 Gene, by Cyclin-dependent Kinase 1Cyclin B THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 51, Issue of ecember 19, pp. 51372-51379, 2003 Aristotelis Astrinidis, William Senapedis, Thomas R. Coleman, and Elizabeth Petri Henske
3.	TSC2: filling the GAP in the mTOR signaling pathway Yong Li, Michael N. Corradetti, Ken Inoki and Kun-Liang Guan
4.	Molecular Cell, Vol. 12, 271-280, August, 2003, Review Integration of Growth Factor and Nutrient Signaling: Implications for Cancer Biology Alykhan F. Shamji, Paul Nghiem,and Stuart L. Schreiber
5.	www.ensembl.org
6.	http://www.bioinfovet.unimi.it/aminoaci.htm 
7.	Tumour suppressors hamartin and tuberin: intracellular signalling. Vera P. Krymskaya. Cellular Signalling 15 (2003) 729–739.  
8.	TSC2 regulates VEGF through mTOR-dependent and -independent pathways. James B. Brugarolas, Francisca Vazquez, Archana Reddy, William R. Sellers, and William G. Kaelin, Jr. CANCER CELL : AUGUST 2003  
9.	Growth Signaling: TSC Takes its Place Steven J. Marygold and Sally J. Leevers. Current Biology, Vol. 12, R785–R787, November 19, 2002.
10.	http://www.dundee.ac.uk/biocentre/SLSBDIV6cgp.htm
11.	http://www.rsc.org/lap/educatio/eic/2002/infochem_nov02.htm
12.	Cell cycle control and cancer. Michelle D. Garrett. CURRENT SCIENCE, VOL. 81, NO. 5, 10         SEPTEMBER 2001
13.	Tuberous sclerosis complex: from Drosophila to human disease. Duojia Pan, Jixin Dong, Yong Zhang and Xinsheng Gao. TRENDS in Cell Biology Vol.14 No.2 February 2004.  
14.	Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Tuberous Sclerosis Tumor Suppressor Complex by Phosphorylation of Tuberin. Han C. Dan, Mei Sun, Lin Yang, Richard I. Feldman, Xue-Mei Sui, Chien Chen Ou, Mark Nellist, Raymond S. Yeung_, Dicky J. J. Halley, Santo V. Nicosia, Warren J. Pledger, and Jin Q. Cheng.  Vol. 277, No. 38, Issue of September 20, pp. 35364–35370, 2003  
15.	The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Hidalgo M, Rowinsky EK. Oncogene. 2000 Dec 27;19(56):6680-6.   
16.	Akt regulates growth by directly phosphorylating Tsc2. Christopher J. Potter, Laura G. Pedraza and Tian Xu. NATURE CELL BIOLOGY VOL 4 SEPTEMBER 2002.
17.	Nutrient-hormone interaction in the ovine liver: methionine supply selectively modulates growth hormone-induced IGF-I gene expression. Stubbs AK, Wheelhouse NM, Lomax MA, Hazlerigg DG. J Endocrinol. 2002 Aug;174(2):335-41.
18.	A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1.
Ozes ON, Akca H, Mayo LD, Gustin JA, Maehama T, Dixon JE, Donner DB. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4640-5. Epub 2001 Apr 03.