History of the concept of TSC

The concept of the tuberous sclerosis complex (TSC), developed over 160 years has come from simple clinical observations, pathological studies and technological advances of imaging methods. It all began with PFO Rayer's color plate of a drawing of a patient who apparently had facial angiofibroma, published in the year 1835, and continued with von Recklinghausen's report of cardiac myomas and cerebral sclerosis in a newborn who had died minutes after birth. The seminal contribution was provided by D.M. Bourneville who, in 1880, reported and named as tuberous sclerosis the neuropathological findings in a young patient with seizures, hemiplegia, and mental subnormality who also had renal tumors. One hallmark of TSC is the presence of tubers: highly epileptogenic dysplastic cerebral cortex composed of abnormally shaped neurons and giant cells. Mutation of the TSC gene (TSC2) may disrupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferation. It is now known that TSC is a hamartomatosis, and thanks to recent studies using positional cloning and DNA analysis, the biological mechanisms underlying these diseases, which include NF1 and NF2 disorders and von Hippel-Lindau disease, are beginning to be understood. Unique to TSC is that it is both phenotypically and genotypically heterogeneous. One of two suspected genes found in chromosome 16 by positional cloning has been cloned (TSC2). The gene product from TSC2 has been named tuberin (1996).

Expression of TSC proteins

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis, a human genetic syndrome characterized by the development of tumors in a variety of tissues. Tuberin is a widely expressed 180 kDa protein that exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. Affinity-purified antibodies against tuberin were used to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression is detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin are found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines reveals a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) reveals that the staining of tuberin is consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolishes the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrates co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tuberin tumor suppressor function (Wienecke, 1996).

Several inherited predispositions to cancer syndromes are associated with the development of nervous system tumors. Tuberous sclerosis complex is an autosomal dominant disorder in which affected individuals are at risk for developing astrocytomas. One of the genes responsible for this disorder is TSC2, located on human chromosome 16p, and encoding a 180 kDa protein (tuberin) that functions in part as a negative regulator of rap1. Thirty percent of sporadic astrocytomas have reduced or absent tuberin expression. In addition to loss of tuberin in sporadic astrocytomas, aberrant rap1 mediated signaling may also result from overexpression of rap1. This study tested the hypothesis that alterations in the rap1 signaling pathway are frequently observed in certain subsets of gliomas compared to other tumors of the nervous system. Analysis of sporadic astrocytomas and ependymomas demonstrates either increased rap1 or reduced/absent tuberin protein expression in 50%-60% of different cohorts of these gliomas, compared to 30%-33% of sporadic schwannomas and meningiomas and none of eight oligodendrocyte tumors. These results suggest that alterations in the rap1 signaling pathway are important in the development of certain sporadic human gliomas (Gutmann, 1997).

Two recently cloned genes produce mutations that result in the phenotype of tuberous sclerosis. TSC2 on chromosome 16p13.3 encodes the protein tuberin, which appears to have growth regulating properties. TSC1 on chromosome 9q34 encodes hamartin, for which to date no cellular functions have been specified. Polyclonal antibodies were raised to synthetic peptides representing portions of tuberin and hamartin and used in immunoblots and immunohistochemical studies to localize the proteins in surgically resected neocortical tubers from four TSC patients. On Western blots of autopsy brain specimens, K-562 cell, and NT2 lysates, each antibody labelled a single band at the expected molecular weight. In immunohistochemical protocols on paraffin embedded tissue, antibodies to both tuberin and hamartin prominently labelled atypical and dysmorphic neuroglial cells that are a defining feature of TSC tubers. Some abnormal cells within cortical tuber sections were labelled with both tuberin and hamartin antisera. These results suggest that tuberin and hamartin are both robustly expressed in similar populations of neuroglial cells of TSC tubers, even in the presence of TSC1 or TSC2 germline mutations. The roles of these gene products in normal and abnormal cortical development, tuber pathogenesis and the generation of seizures remain to be defined (Johnson, 1999).

Mapping and characterization of TSC genes

Linkage studies have shown locus heterogeneity with a TSC gene mapped to chromosome 9q34 and a second gene, recently identified on 16p13.3. DNA markers have been analyzed in eight hamartomas and one tumour from TSC patients: allele loss on 16p13.3 has been found in three angiomyolipomas, one cardiac rhabdomyoma, one cortical tuber and one giant cell astrocytoma. It is suggested that the TSC gene on 16p13.3 functions like a tumour suppressor gene, in accordance with Knudsen's hypothesis (Green, 1994).

32 families informative for the segregation of Tuberous sclerosis have been examined for genetic markers on chromosomes 9, 11, 12 and 16. In one large family there was clear evidence of linkage to markers on chromosome 16p13.3 (lodscore with D16S291 of 4.7 at theta = 0) but other families were too small to give individually convincing lodscores. Combined results for all families gave positive results with ABO/DBH on chromosome 9 (max lod 2.63) and with D16S291 on chromosome 16 (max lod 3.98) at theta values of 0.2 in each case. Further analysis showed strong evidence for heterogeneity with approximately half the families linked to a locus TSC1 on chromosome 9 between ASS and D9S298 and half to TSC2 on chromosome 16 close to D16S291. There was no definite support for a third locus although in many families this could not be excluded. In three families the segregation pattern of TSC remains unexplained. In two of these the family apparently segregates for TSC1 but in each case a single affected individual appeared to exclude the whole of the candidate region. Preliminary analysis of clinical features did not reveal any definite differences in incidence of mental handicap between individuals in different linkage groups or with the sex of the parent of origin. The frequencies of periungual fibromas and facial angiofibromas were also similar in both linkage groups (Povey, 1994).

TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor (van Slegtenhorst, 1997).

Functions and interactions of TSC proteins and alternative splicing of TSC mRNAs

Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. Antisera against the N-terminal and C-terminal portions of tuberin were produced. These antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation reveals that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin does not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis (Wienecke, 1995).

The Eker rat hereditary renal carcinoma is an excellent example of Mendelian dominant predisposition to a specific cancer in an experimental animal. A germline insertion in the rat homolog of the human tuberous sclerosis gene (TSC2) gives rise to dominantly inherited cancer in the Eker rat model, as well as a tumor suppressor nature for the Tsc2 gene function. There is a strong conservation between the rat and human gene products. The molecular function of the Tsc2 gene product (called 'tuberin' in the human case) is not yet understood, although it contains a short amino acid sequence homologous to ras family GTPase-activating proteins (Rap1GAP). Transcriptional activation domains (AD1 and AD2) in the carboxyl terminus of the Tsc2 product (in exons 30 and 32 and exon 41, respectively) are described. The Eker insertional mutation (intron 30) disrupts transcriptional activity. Whereas a COOH-terminal truncated Tsc2 protein was localized in the nucleus, the full-length protein is found predominantly in the perinuclear region of cytoplasm. The present demonstration of transcriptional activation domains in the Tsc2 gene provides clues for studying its role in renal carcinogenesis (Tsuchiya, 1996).

Expression of the wild-type TSC2 gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This 'suppressor' activity is encoded by a functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2 (a cytosolic factor, rabaptin-5) was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possess activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity is specific for Rab5 and shows no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possess minimal Rab5GAP activity and are associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduces the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis (Xiao, 1997).

TSC2 encodes tuberin, a putative GTPase activating protein for rap1 and rab5. The TSC1 gene was recently identified and codes for hamartin, a novel protein with no significant homology to tuberin or any other known vertebrate protein. Hamartin and tuberin are shown to associate physically in vivo and the interaction is mediated by predicted coiled-coil domains. These data suggest that hamartin and tuberin function in the same complex rather than in separate pathways (van Slegtenhorst, 1998).

Tuberous sclerosis is a genetic disorder that results in the development of hamartomatous lesions in a variety of organ systems. Both the prevalence of the disease and the often devastating consequences of these tumors pose a serious health and medical care problem. The disease has been mapped to two distinct genetic loci in humans, and although the genes (TSC1 and TSC2) for both loci have recently been cloned, their function remains an enigma. Data presented here demonstrates that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes. These data place TSC2 into a growing list of nuclear receptor coregulators and strengthen the expanding body of evidence that these coregulators may play critical roles in cellular differentiation (Henry, 1998).

The gene for tuberous sclerosis 2 (TSC2) encodes a 5.5-kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. A novel variant of the TSC2 mRNA lacking 129 nucleotides was detected, predicting an in-frame deletion of 43 amino acids spanning codons 946-988 of tuberin. This 129-bp deletion precisely corresponds to exon 25 of the TSC2 gene suggesting that alternative splicing leads to production of two forms of transcripts designated isoforms 1 and 2. Further molecular analysis reveals a third isoform exhibiting a deletion of 44 amino acids spanning codons 946-989 of tuberin. Amino acid 989 is a Ser residue encoded by the first codon of exon 26. The two isoforms also exist in newborn and adult mouse tissues, reinforcing the potential functional importance of these alternatively spliced products. These alternative isoforms should have implications for efforts aimed at identifying mutations in TSC patients. The distinct polypeptides encoded by the TSC2 gene may have different targets as well as functions involved in the regulation of cell growth (Xu, 1995).

TSC gene mutation and phenotypic effects

Angiomyolipomas (AMLs) are renal tumors that occur both sporadically and in association with tuberous sclerosis. TSC is an autosomal dominant disorder characterized by hamartomatous lesions in multiple organs. Two TSC loci are recognized: TSC1 on 9q34 and TSC2 on 16p13. Loss of heterozygosity (LOH) at the TSC1 and TSC2 loci in lesions from TSC patients has recently been reported. Lesions that are not associated with TSC have not been previously examined for LOH at the TSC loci. This study 29 renal angiomyolipomas from patients without a history of TSC. Three tumors demonstrated LOH on 16p13. This is the first report indicating that mutations in TSC2 occur in tumors of patients who do not have TSC. LOH was also found on 16p13 in 5 of 8 TSC-associated AMLs. Two of these tumors were from a single patient and demonstrated different regions of LOH. These findings support the hypothesis that the TSC2 gene functions as a tumor suppressor (Henske, 1995).

Linkage studies have shown locus heterogeneity with one TSC gene mapped to chromosome 9q34 and a second to 16p13.3. The gene on 16p13.3, TSC2, has been cloned and shown to encode a 5.5 kb transcript that is widely expressed. To facilitate the search for mutations in the TSC2 gene product, tuberin, an RT-PCR-based assay system has been designed to scan the expressed coding region of the TSC2 gene in lymphoblasts. Using 34 overlapping PCR assays single-strand conformation polymorphism analysis was performed of DNA from 26 apparently sporadic TSC cases, two TSC families non-informative for linkage analysis and two confirmed chromosome 16-linked TSC families. Of the 60 chromosomes scanned, 14 showed abnormal SSCP mobility shifts. Using direct PCR sequencing five missense mutations, one 3 bp in-frame deletion and one 2 bp frameshift deletion, one nonsense mutation, one 29 bp tandem duplication and five silent nucleotide changes that are likely to be polymorphisms have been identified. There is no apparent clustering of mutations within TSC2. The diversity of mutation types argues that TSC2 may not act in a classic tumor suppressor fashion. In addition, no specific correlation was seen between the different mutations and clinical severity or expression. These data confirm that TSC2 is indeed the relevant gene, and that a substantial number of sporadic cases arise from mutations in the TSC2 gene (Wilson, 1996).

Mutation of the TSC gene (TSC2) may disrupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferation. To test the hypothesis that cells in tubers may retain the molecular phenotype of embryonic or immature neurons, tubers from five TSC patients were probed with antibodies to proteins expressed in neuronal precursors (nestin, Ki-67, and proliferating cell nuclear antigen). Many dysmorphic neurons and giant cells in tubers were stained by these antibodies, while neurons in adjacent normal and control cortex were not labeled. To further characterize the molecular phenotype of cells in tubers, a methodology was developed in which poly(A)+ mRNA is amplified from immunohistochemically labeled single cells in paraffin-embedded brain specimens. This approach enabled the detection of mRNAs encoding nestin, and other cytoskeletal elements, cell cycle markers, and synthetic enzymes present in individual nestin-stained cells by means of reverse Northern blotting. It is concluded that the presence of immature phenotypic markers (mRNAs and proteins) within tubers suggests disruption of cell cycle regulation and neuronal maturation in TSC during cortical development. Characterization of multiple mRNAs within fixed, immunohistochemically labeled cells provides a powerful tool for studying gene expression and the molecular pathophysiology of many neurologic diseases (Crino, 1996).

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. The homozygous state of this mutation [Tsc2(Ek/Ek)] is lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA is highly expressed in embryonic neuroepithelium. During this period, homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, display dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupts the normal development of this tissue. Interestingly, there is significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos display these defects whereas the Fisher 344 homozygous mutant embryos have normal-appearing neuroepithelium. Taken together, these data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain (Rennebeck, 1998).

Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis, a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, a Tsc2 mutant mouse was generated. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants die around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibit an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis (Kobayashi, 1999).

It has been shown that the genes responsible for TSC, TSC1 and TSC2, act as tumor suppressors, but the mechanism of hamartomatous growth in several tissues is not completely understood. The TSC hamartomas are essentially benign and they rarely progress to malignant tumors. The angiofibroma stroma cells of three adult TSC patients were cultured and these cells were compared with normal skin fibroblasts for their proliferative capacity, cell morphology and mitotic cycle using a stain for microtubules and the expression of the senescent associated beta-galactosidase (SA beta-Gal). Cultured angiofibroma stroma cells from TSC patients displayed several characteristics observed in human senescent fibroblasts: a low proliferative capacity, an increase in cell size, increased binucleated cells in association with abnormal cytokinesis and increased SA beta-Gal positives. Growth of facial angiofibromas in TSC may be caused by a gain in enhanced sensitivity toward some of the potential mitogens and forced multiplication without loss of the cellular senescent program; this may be the reason why TSC hamartomas rarely progress to malignancy and why the growths are limited to a finite size (Toyoshima, 1999).

Tuberous sclerosis (TSC) is a autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, and characterized by benign hamartoma growth. A murine model of Tsc1 disease was developed by gene targeting. Tsc1 null embryos die at mid-gestation from a failure of liver development. Tsc1 heterozygotes develop kidney cystadenomas and liver hemangiomas at high frequency, but the incidence of kidney tumors is somewhat lower than in Tsc2 heterozygote mice. Liver hemangiomas are more common, more severe and cause higher mortality in female than in male Tsc1 heterozygotes. Tsc1 null embryo fibroblast lines have persistent phosphorylation of the p70S6K (S6K) and its substrate S6; this phosphorylation is sensitive to treatment with rapamycin, indicating constitutive activation of the mTOR-S6K pathway due to loss of the Tsc1 protein, hamartin. Hyperphosphorylation of S6 is also seen in kidney tumors in the heterozygote mice, suggesting that inhibition of this pathway may have benefit in the control of TSC hamartomas (Kwaitkowski, 2002).

The role of tumor suppressor haploinsufficiency in oncogenesis is still poorly understood. The PTEN and TSC2 tumor suppressors function to antagonize mTOR (mammalian target of rapamycin) activation by Akt; hence, compound heterozygous inactivation of Pten and Tsc2 in the mouse may in principle exacerbate the tumor phenotypes observed in the single mutants in a reciprocal manner. In contrast, it was found that while Tsc2 heterozygosity unmasks Pten haploinsufficiency in growth and tumor suppression, tumorigenesis in Tsc2+/- mutants is surprisingly not accelerated by Pten heterozygosity, even though mTOR activation is cooperatively enhanced by compound Pten/Tsc2 heterozygosity. The wild-type alleles of both Pten and Tsc2 are retained in prostate tumors from both Pten+/- and Pten+/-Tsc2+/- mice, whereas TSC-related tumor lesions are invariably associated with Tsc2 loss of heterozygosity (LOH) in both Tsc2+/- and Pten+/-Tsc2+/- mice. These findings demonstrate that inactivation of TSC2 is epistatic to PTEN in the control of tumor initiation and progression and, importantly, that both Pten and Tsc2 are haploinsufficient for suppression of tumorigenesis initiated by Pten heterozygosity, while neither Pten nor Tsc2 is haploinsufficient for repression of carcinogenesis arising from Tsc2 heterozygosity, providing a rationale for the differential cancer susceptibility of the two human conditions associated with PTEN or TSC2 heterozygous mutations (Ma, 2005).

The PTEN and TSC2 tumor suppressors inhibit mammalian target of rapamycin (mTOR) signaling and are defective in distinct hamartoma syndromes. Using mouse genetics, it has been found that Pten and Tsc2 act synergistically to suppress the severity of a subset of tumors specific to loss of each of these genes. Interestingly, the slow-growing tumors specific to Tsc2+/- mice exhibit defects in signaling downstream of Akt. However, Pten haploinsufficiency restores Akt signaling in these tumors and dramatically enhances their severity. This study demonstrates that attenuation of the PI3K-Akt pathway in tumors lacking TSC2 contributes to their benign nature (Manning, 2005).

Axon formation is fundamental for brain development and function. TSC1 and TSC2 are two genes, mutations in which cause tuberous sclerosis complex (TSC), a disease characterized by tumor predisposition and neurological abnormalities including epilepsy, mental retardation, and autism. This study shows that Tsc1 and Tsc2 have critical functions in mammalian axon formation and growth. Overexpression of Tsc1/Tsc2 suppresses axon formation, whereas a lack of Tsc1 or Tsc2 function induces ectopic axons in vitro and in the mouse brain. Tsc2 is phosphorylated and inhibited in the axon but not dendrites. Inactivation of Tsc1/Tsc2 promotes axonal growth, at least in part, via up-regulation of neuronal polarity SAD kinase, which is also elevated in cortical tubers of a TSC patient. The results reveal key roles of TSC1/TSC2 in neuronal polarity, suggest a common pathway regulating polarization/growth in neurons and cell size in other tissues, and have implications for the understanding of the pathogenesis of TSC and associated neurological disorders and for axonal regeneration (Choi, 2008).

Axon formation in the brain is governed by extracellular signals and the intrinsic polarization apparatus. Tsc1/Tsc2 is ideally suited to couple extracellular signals to the intracellular growth machinery. It was observed that Tsc2 is phosphorylated, and thus inhibited, and the mTORC1 pathway is activated in the axon. Inactivation of Tsc1/Tsc2 allows the prospective axon to initiate and maintain its growth through mTORC1, while Tsc1/Tsc2 keeps the growth of the remaining neurites/dendrites on hold. Growth factors such as IGF-1 (insulin-like growth factor-1) and BDNF (brain derived growth factor) activate PI3K and Akt, which phosphorylate and inactivate Tsc2. Furthermore, Wnt inhibition of GSK3, which phosphorylates and activates Tsc2, also leads to Tsc2 inactivation. Thus Tsc2 regulation may mediate the capability of IGF, BDNF, and Wnt in specifying neuronal polarity and axonal morphogenesis. It has been suggested that Akt promotes axon formation by phosphorylation and inhibition of GSK3. However, mice harboring GSK3 knockin alleles that abolish the Akt phosphorylation site are viable and fertile, and cortical and hippocampal neurons of these mice are polarized normally in vitro and in vivo. These results together suggest that Akt phosphorylation of Tsc2, but not GSK3, likely plays a critical role in neuronal polarity/axonal growth (Choi, 2008 and references therein).

Signaling upstream of TSC2

Tuberous sclerosis complex (TSC) and Peutz-Jeghers syndrome (PJS) are dominantly inherited benign tumor syndromes that share striking histopathological similarities. LKB1 (Drosophila homolog: lkb1), the gene mutated in PJS, acts as a tumor suppressor by activating TSC2, the gene mutated in TSC. Like TSC2, LKB1 inhibits the phosphorylation of the key translational regulators S6K and 4EBP1. Furthermore, LKB1 activates TSC2 through the AMP-dependent protein kinase (AMPK: Drosophila homolog SNF1A/AMP-activated protein kinase), indicating that LKB1 plays a role in cell growth regulation in response to cellular energy levels. These results suggest that PJS and other benign tumor syndromes could be caused by dysregulation of the TSC2/mTOR pathway (Corradetti, 2004).

Peutz-Jeghers syndrome (PJS), a dominantly inherited genetic disorder, is characterized by the formation of gastrointestinal hamartomas that are histologically similar to those observed in TSC patients. PJS is associated with mutations in the lkb1 tumor suppressor gene, which codes for a serine/threonine kinase. Although extensive work has been performed on the molecular pathogenesis of TSC, the molecular mechanism of LKB1 as a tumor suppressor has remained elusive (Corradetti, 2004 and references therein).

Recent studies from several laboratories have demonstrated that LKB1 phosphorylates and activates AMPK, representing the first convincing physiological target of LKB1. AMPK is a multimeric protein, and its kinase activity is enhanced by both phosphorylation and high intracellular AMP levels. The amount of AMP in the cell is inversely proportional to the amount of ATP, and high levels of AMP are present under low energy conditions. Under such conditions, AMPK is activated and phosphorylates numerous substrates to suppress anabolism and enhance catabolism, thereby regulating cellular energy homeostasis. LKB1 potentiates the effect of AMP on AMPK by phosphorylating AMPK on Thr 172, a residue found in the AMPK activation loop (Corradetti, 2004 and references therein).

Activated AMPK phosphorylates and activates the TSC2 tumor suppressor protein. This AMPK-dependent regulation of TSC2 is especially important for cellular energy response because cells expressing TSC2 mutants that cannot be phosphorylated by AMPK undergo apoptosis under energy starvation conditions. TSC2 displays GTPase activating protein (GAP) activity toward the small G-protein Rheb (Ras homolog enriched in brain), and both biochemical and genetic studies have established that TSC2 acts through Rheb to inhibit mTOR function. Recent studies have also found that mTOR activation is important for the secretion of vascular endothelial growth factor (VEGF), which is a potent prooncogenic factor that increases the growth of blood vessels near tumors. Consistent with this observation, VEGF secretion is enhanced in TSC2-/- cells, and rapamycin effectively inhibits the secretion of VEGF from these cells. LKB1-/- cells are also reported to secrete high levels of VEGF compared with their cognate wild-type cells. Given the biochemical relationship between LKB1 and AMPK, the observation that LKB1-/- and TSC2-/- cells display high VEGF expression, and the histological similarity between the hamartomas of PJS and TSC, the functional relationship between the two tumor suppressors, LKB1 and TSC2, was examined (Corradetti, 2004).

LKB1 is one of the first protein kinases shown to function as a tumor suppressor. Despite extensive biochemical, genetic, and cell biological studies, the molecular mechanism of LKB1 as a tumor suppressor has remained elusive. However, the identification of AMPK as a physiological substrate of LKB1 and the observations described in this report provide an important clue for how LKB1 could negatively regulate cell growth. The following mechanism is proposed to explain how LKB1 activates the TSC2 tumor suppressor: LKB1 directly phosphorylates and activates AMPK. The active AMPK then phosphorylates TSC2 to enhance TSC2 function. TSC2 subsequently inhibits mTOR function via TSC2's GAP activity toward the Rheb small GTPase. Therefore, it is postulated that LKB1 negatively regulates cell growth by inhibiting the phosphorylation of important mTOR targets such as S6K and 4EBP1. It should be noted that, in addition to AMPK, LKB1 can phosphorylate and activate several AMPK-related kinases—although these kinases are not regulated by AMP. It remains to be seen whether these AMPK-related kinases can also contribute to LKB1-mediated phosphorylation of TSC2. However, the results make clear that the LKB1-AMPK kinase cascade likely plays a major role in TSC2-mTOR regulation of cellular energy response. Moreover, these data indicate that mutation in TSC2 or LKB1 produces similar cellular phenotypes (rapamycin-sensitive apoptosis and VEGF production), supporting the idea that the two tumor-suppressor proteins function in the same pathway (Corradetti, 2004).

It should also be noted that mutations in the phosphoinositide phosphatase PTEN are associated with Cowden's disease and the Bannayan-Riley-Ruvalcaba syndrome, two other dominantly inherited hamartoma syndromes. Loss of PTEN leads to an increase in 3-phosphoinositide concentration and subsequent activation of AKT, and AKT has been shown to inhibit TSC2 function. Thus, the pathophysiology of several syndromes associated with benign tumors may converge on the TSC2 and mTOR pathway. It is also especially significant that mTOR is a key downstream target of LKB1 in the regulation of cell growth, because the immunosuppressant drug rapamycin specifically inhibits mTOR. Recently, murine studies and clinical trials have indicated that rapamycin and other mTOR inhibitors may be potent drugs for the treatment of TSC and other neoplasms. These studies suggest that rapamycin and mTOR inhibitors may also be potential drugs for the comprehensive treatment of PJS (Corradetti, 2004).

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. This study shows that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801 (Drosophila homologs: scylla and charybde). Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia (Brugarolas, 2004).

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. This study shows that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK; see Drosophila RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, these data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function (Roux, 2004).

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. The stress response REDD1 gene has been identified as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, because regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway (Sofer, 2005).

Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling

Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Tuberous sclerosis 1 (Tsc1) and Tsc2, tumor suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. It is proposed that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. These studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumor suppressors (Gao, 2002).

TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. These data indicate a molecular mechanism for TSC2 in insulin signalling, tumor suppressor functions and in the inhibition of cell growth (Inoki, 2002).

TSC proteins control mTOR signaling by acting as a GTPase-Activating Protein Complex toward Rheb

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).

The tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product is a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, whether a Ras/Rap-related GTPase might be involved in this process was examined. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promotes the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation is also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug (Castro, 2003).

Tuberous sclerosis complex is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. These data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth (Inoki, 2003a).

The small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1; see Drosophila S6kII) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, it is shown that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. It is concluded that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells (Tee, 2003).

To examine whether Rheb overexpression could modulate S6K1 activity, S6K1 was coexpressed with Rheb at two different expression levels in HEK293E cells and kinase activity was assayed by using GST-S6 as a substrate. Coexpression of Rheb significantly increased the basal and insulin-stimulated activity of S6K1. Higher levels of Rheb expression enhanced the basal and insulin-stimulated activity of S6K1 by 5.6- and 1.7-fold, respectively, and this activity level is more potent than the S6K1 activity observed in the presence of lower levels of Rheb protein. These results indicate that Rheb activates signaling cascades that result in S6K1 activation (Tee, 2003).

Given that the activity of S6K1 is enhanced upon cell signaling through mTOR, PI3K, and mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)-mediated pathways, it was determined which signaling pathway was activated upon Rheb overexpression. To investigate PI3K-mediated signaling, Rheb, along with Akt, a downstream target of PI3K, was expressed within HEK293E cells and kinase activity was assayed. Whereas EGF stimulation led to a 4-fold increase in Akt activity, Rheb overexpression did not enhance basal or EGF-stimulated Akt activity. In contrast, Rheb potently activated S6K1 by 11-fold when assayed in parallel. Wortmannin was used as a control to show that PI3K-mediated activation of Akt upon stimulation with EGF was specifically measured. To examine whether Rheb enhanced MAPK-mediated signaling, Rheb was coexpressed with RSK1, a known downstream signaling component of MAPK, within HEK293E cells. Although EGF stimulation led to a 12-fold increase in RSK1 activity, Rheb did not augment the basal or EGF-induced activation of RSK1 assayed with GST-S6 as a substrate. In contrast, Rheb overexpression drastically increased S6K1 activity when assayed in parallel. To inhibit activation of ERK (extracellular signal-regulated kinase) through MEK (MAPK/ERK-kinase)-mediated signaling, cells were treated with the U0126 compound to specifically inhibit MEK. These findings suggest that Rheb does not function upstream of either PI3K/Akt or ERK/RSK1 signaling pathways (Tee, 2003).

Because Rheb enhances the activity of S6K1, a downstream component of mTOR, the effects were investigated of Rheb-mediated signaling on 4E-BP1, another downstream component of mTOR. Dephosphorylated species of 4E-BP1 bind to and inhibit eIF4E-driven cap-dependent translation. Phosphorylation of 4E-BP1 at multiple Ser/Thr-Pro residues upon mitogenic stimulation led to the release of 4E-BP1 from eIF4E, which is blocked by both rapamycin and nutrient starvation. Three different phosphorylated species of 4E-BP1 resolve on SDS-PAGE, with gamma- and alpha-isoforms being the most and least phosphorylated species, respectively. To determine whether Rheb activates mTOR- or PI3K-mediated signaling, Rheb was coexpressed with hemagglutinin (HA)-tagged 4E-BP1 in the presence of either rapamycin or wortmannin to inhibit mTOR or PI3K, respectively. Insulin-induced Akt phosphorylation on Ser473 was blocked by wortmannin, revealing that the concentration of wortmannin used in this study efficiently inhibits PI3K-mediated signaling. Insulin-induced phosphorylation of 4E-BP1 was also blocked by wortmannin, as observed by the reduced mobility shift of 4E-BP1 to the less-phosphorylated isoforms and by decreased Ser65 phosphorylation. Rheb overexpression within serum-starved cells potently enhances 4E-BP1 phosphorylation, which was still sensitive to rapamycin. In contrast, treatment of cells with wortmannin was modestly effective at reducing 4E-BP1 phosphorylation upon Rheb overexpression, indicating that PI3K signaling is not essential for Rheb-induced 4E-BP1 phosphorylation. These data suggest that Rheb signals by using an mTOR-dependent rather than a PI3K-dependent mechanism. Rheb-induced 4E-BP1 phosphorylation should promote the release of 4E-BP1 from eIF4E. To confirm this, endogenous eIF4E was purified on m7GTP-Sepharose, which mimics the cap-structure found at the extreme 5' terminus of most cytoplasmic mRNAs, and how much HA-tagged 4E-BP1 was bound to eIF4E was examined. As expected, 4E-BP1 is released from eIF4E upon Rheb overexpression, implying that Rheb activates cap-dependent translation (Tee, 2003).

The above data implies that Rheb may modulate mTOR signaling. Given that the loss of Rheb in yeast mimics nutrient starvation, Rheb overexpression may promote mTOR signaling through a nutrient-regulated signaling pathway. To address this possibility, whether Rheb promotes S6K1 activation was investigated in the absence of amino acids. During conditions of amino acid withdrawal, Rheb overexpression potently activated S6K1, which was completely blocked by rapamycin but only partially inhibited by wortmannin. Importantly, insulin stimulation of these amino acid-deprived cells potently activated Akt (as observed by Akt phosphorylation on Ser473) but only weakly activated S6K1. Therefore, unlike Rheb-mediated signaling, acute stimulation of PI3K and Akt is not sufficient to fully activate S6K1 during nutrient insufficiency. The modest insulin-induced activation of S6K1 during amino acid insufficiency was blocked by wortmannin, revealing that this activation is completely dependent on PI3K. Activation of S6K1 upon readdition of amino acids was enhanced when Rheb was overexpressed or when cells were stimulated with insulin). Interestingly, Rheb-induced S6K1 activation upon readdition of amino acids was completely inhibited by rapamycin but only partially inhibited by wortmannin. In contrast, insulin-induced S6K1 activity was markedly impaired by both rapamycin and wortmannin. These data convincingly reveal that Rheb potently activates S6K in the absence of nutrients through mTOR rather than PI3K-mediated signaling. Therefore, it is likely that Rheb enhances nutrient-mediated signaling through mTOR (Tee, 2003).

To decisively determine whether Rheb positively activates mTOR signaling, use was made of a rapamycin-resistant mutant of S6K1 (S6K1-F5A-DeltaCT). Unlike treatment with wortmannin, rapamycin treatment was unable to prevent insulin-induced activation of S6K1-F5A-DeltaCT, demonstrating that this mutant is responsive to PI3K signaling but not mTOR signaling. As a positive control, PDK1 and PKCζ, which are known to activate S6K1 through a PI3K-dependent input, were coexpressed. Overexpression of Rheb potently activates wild-type S6K1 basally (by 11-fold) and during insulin stimulation (by 17-fold) but does not enhance the activity of the S6K1-F5A-DeltaCT mutant. In contrast, increased PI3K-mediated signaling toward S6K1 by coexpression of PDK1 and PKCζ results in significantly enhanced activation of both wild-type S6K1 and S6K1-F5A-DeltaCT. These findings strongly suggest that Rheb induces S6K1 activation via a signaling input that is upstream of mTOR but not PI3K (Tee, 2003).

Overexpression of wild-type Rheb has been shown to lead to a significant increase in its activity and implies that the majority of the overexpressed Rheb must exist in the active GTP bound form. If this is true, then the RhebGAP activity must be a limiting factor. If Tuberin possesses RhebGAP activity, overexpression of Tuberin should switch Rheb from an active GTP bound state to an inactive GDP bound state. To indirectly measure Rheb activity, Rheb-induced S6K1 activation was analyzed within nutrient-deprived HEK293E cells. Coexpression of Hamartin and Tuberin completely block Rheb's ability to activate S6K1, implying that Tuberin may function as a RhebGAP (Tee, 2003).

If the GAP domain of Tuberin is essential for Rheb inactivation, then patient-derived TSC2 GAP domain point mutants should not block Rheb-induced S6K1 activation. To address this, three Tuberin mutants were generated that mimic patient-derived TSC2 mutations that occur within the GAP domain and these Tuberin mutants were coexpressed with Hamartin and S6K1. Under serum-starved conditions, Rheb potently activates S6K1, which was fully blocked by coexpression of wild-type Tuberin with Hamartin. In contrast, the three TSC2 GAP domain point mutants were unable to repress Rheb-induced S6K1 activation, revealing that the GAP domain of Tuberin is critical for Tuberin's ability to repress Rheb-mediated signaling (Tee, 2003).

The in vivo overexpression data strongly suggest that the Tuberin-Hamartin heterodimer inhibits Rheb function. In order to test whether this is a direct inhibition due to the GAP activity of Tuberin, in vitro GAP assays were performed on purified Rheb. Flag-tagged Hamartin and Flag-tagged Tuberin were expressed separately or together in HEK293 cells and the respective protein(s) were immunoprecipitated for use in Rheb-GAP assays. Interestingly, immunoprecipitated Hamartin or Tuberin display nearly identical GAP activity toward Rheb; both enhance the intrinsic GTPase activity of Rheb by approximately 2-fold. This suggests that a complex between Tuberin and Hamartin is essential for Tuberin's GAP activity toward Rheb and that endogenous levels of coimmunoprecipitating Tuberin or Hamartin are limiting in these reactions. In support of this, coexpression and immunoprecipitation of both Tuberin and Hamartin result in immune complexes that enhance Rheb GTPase activity by more than 100-fold over the activity of either alone and approximately 200-fold over intrinsic Rheb activity. This dramatic increase in GAP activity is detected despite no significant difference in the amount of Tuberin immunoprecipitated when expressed alone or with Hamartin. Therefore, Tuberin and Hamartin together form a GTPase-activating protein complex that greatly enhances the intrinsic GTPase activity of Rheb (Tee, 2003).

In order to test if this activity is potentially important in the prevention of the TSC disease, the GAP activity of wild-type Tuberin was compared to that of a patient-derived mutant mapped to the Tuberin GAP domain (N1651S). Compared to wild-type Tuberin, the Tuberin(N1651S) mutant is greatly reduced in its ability to enhance Rheb GTPase activity. This suggests that there is a correlation in the ability of Tuberin to act as a GAP toward Rheb and its ability to suppress the TSC disease (Tee, 2003).

Farnesylation of Rheb is required for cell cycle progression of S. pombe. To investigate whether farnesylation is important for Rheb's ability to activate S6K1, a farnesylation-defective Rheb(C182S) mutant was generated, in which the cysteine within the farnesylation CAAX motif is substituted for a serine. When overexpressed, the Rheb(C182S) mutant is less efficient at enhancing S6K1 activity than wild-type Rheb. The mutant Rheb(C182S) protein migrated as the upper band on SDS-PAGE, which indicates that it is not being prenylated and is consistent with earlier studies showing that prenylated Rheb migrates more quickly on SDS-PAGE. In contrast, the majority of wild-type Rheb resolved as the lower prenylated band. These findings suggest that the membrane localization of Rheb through farnesylation is important for Rheb to efficiently augment mTOR-mediated signaling (Tee, 2003).

Thus, Rheb functions upstream of mTOR within the nutrient signaling pathway. Rheb specifically activates mTOR-mediated signaling rather than cell signaling through MEK/ERK and PI3K, as shown by Rheb-mediated activation of S6K1 but not Akt or RSK1. Therefore, it is unlikely that Rheb activates PI3K and Raf, two downstream effectors of Ras. Rheb has previously been shown to interact with Raf in vitro, but the current data suggest that Raf is not an effector of Rheb in vivo. Additionally, Rheb overexpression does not increase the activity of the rapamycin-resistant S6K1 mutant that is unresponsive to mTOR signaling inputs but is activated in response to PI3K signaling. S6K1 activation is regulated by multiple signaling inputs, one of which is directed by PI3K. Therefore, these findings are important and confirm that Rheb overexpression specifically promotes mTOR rather than PI3K signaling. Furthermore, Rheb-induced 4E-BP1 phosphorylation is completely sensitive to rapamycin but not to wortmannin, which further strengthens the notion that Rheb acts upstream of mTOR rather than PI3K. 4E-BP1 dissociates from eIF4E upon Rheb overexpression, revealing that Rheb-mediated signaling through mTOR promotes cap-dependent translation (Tee, 2003).

Evidence has also been provided that Rheb functions within the nutrient signaling cascade upstream of mTOR, as shown by Rheb's ability to potently stimulate S6K1 activity during amino acid insufficiency. During amino acid withdrawal, acute insulin stimulation is still able to elicit high levels of Akt phosphorylation but poorly activates S6K1, showing that the nutrient-mediated mTOR signaling input is essential for optimal S6K1 activation. Therefore, Rheb overexpression supersedes the dependency of the nutrient input to mTOR, suggesting that Rheb is an activator of mTOR within the nutrient-signaling pathway. Interestingly, resupplying cells with amino acids further enhances the activity of S6K1 when Rheb is overexpressed, suggesting that amino acids may promote the activation of Rheb. This research has revealed that the Tuberin-Hamartin heterodimer functions as an inhibitor of nutrient signaling through mTOR. The Tuberin-Hamartin heterodimer inhibits Rheb-induced S6K1 activation during conditions of amino acid withdrawal. This work, therefore, extends these earlier studies, revealing that inhibition of Rheb is the mechanism by which the Tuberin-Hamartin heterodimer inhibits nutrient-mediated signaling. Importantly, the Rheb-inhibitory function of Tuberin-Hamartin heterodimers depends on an intact Tuberin GAP domain; patient-derived point mutations within the GAP domain of TSC2 prevented the Tuberin-Hamartin heterodimer from blocking Rheb-induced S6K1 activation. These data indicate that the GAP activity of Tuberin promotes inactivation of Rheb in vivo, presumably through increasing the intrinsic GTPase activity of Rheb. Confirming this hypothesis, in vitro Rheb GTPase activity assays have revealed that Tuberin enhances the intrinsic GTPase activity of Rheb. Interestingly, coexpression of Hamartin with Tuberin markedly enhances the GTPase activity of Rheb, implying that Hamartin promotes the GAP function of Tuberin toward Rheb. A model is proposed whereby Rheb promotes mTOR signaling when it is in an active GTP bound form, whereas the Tuberin-Hamartin heterodimer inhibits Rheb by converting it to an inactive GDP bound state. These findings reveal that the Tuberin-Hamartin heterodimer and Rheb respectively inhibit and activate the nutrient-signaling input to mTOR. Small G proteins are additionally regulated by guanine nucleotide exchange factors (GEFs). In this model it is proposed that a RhebGEF becomes activated during conditions of nutrient sufficiency, and its activation switches Rheb to an active GTP bound form. Therefore, identifying this Rheb-GEF will be of great importance and may provide new insights into how mTOR senses intracellular amino acids. However, at this point the possibility that Rheb- and nutrient-mediated signaling may function in parallel pathways upstream of mTOR cannot be ruled out. Further experiments will be carried out to investigate this possibility (Tee, 2003).

Akt targets TSC2

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB (see Drosophila Akt1). Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).

Class I phosphoinositide 3-kinases (PI3Ks) are activated by many extracellular growth and survival stimuli. These lipid kinases catalyze the production of the second messengers phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5P3). Downstream targets containing specialized domains, such as pleckstrin-homology (PH) domains, that specifically bind to these lipid products of PI3K are then activated. These activated proteins control a wide array of cellular processes, including survival, proliferation, protein synthesis, growth, metabolism, cytoskeletal rearrangements, and differentiation. However, there is still much that is not known about the signaling events leading from activation of PI3K effectors to downstream changes in cell physiology (Manning, 2002).

Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).

The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).

Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by the current knowledge of downstream targets (Manning, 2002).

An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite ( Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).

TSC is a common disease affecting an estimated 1 in 6000 individuals and is characterized by the occurrence of widespread benign tumors called hamartomas frequently affecting the brain, skin, kidneys, lungs, eyes, and heart. In approximately 85% of TSC patients, the disease is caused by loss-of-function mutations in one of two tumor suppressor genes, TSC1 and TSC2, which encode hamartin and tuberin, respectively. These two proteins form a complex. Tuberin, which has a region of homology to Rap1 GTPase-activating proteins (GAPs), has been shown to possess in vitro GAP activity toward Rap1. However, the true molecular and cellular functions of the hamartin-tuberin complex have yet to be clearly defined. Furthermore, very little is known about how these tumor suppressor gene products are regulated (Manning, 2002).

Based on genetic studies and the fact that PI3K and Akt are oncogenes while the TSC genes are tumor suppressors, one would predict that the PI3K-Akt-mediated phosphorylation of tuberin would inhibit the function of the tuberin-hamartin complex. In Drosophila, hamartin and tuberin appear to function together to antagonize signaling of the insulin-PI3K-Akt pathway and, thereby, restrict cell growth and proliferation. Most strikingly, loss of just one copy of TSC1 or TSC2 partially rescues the lethality of insulin receptor loss-of-function mutants. This result implies that one of the primary functions of the insulin pathway, at least in Drosophila, is to inhibit the hamartin-tuberin complex. Furthermore, both mouse and Drosophila genetic studies have suggested that the tuberin-hamartin complex functions to inhibit S6K1 (Manning, 2002).

Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).

Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).

The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).

Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. These results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function (Dan, 2002).

The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B) regulates cell growth is unknown. Drosophila Akt/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1-Tsc2 complex. Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved residues, Ser 924 and Thr 1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, increasing the stability of the Tsc1-Tsc2 complex within the cell. Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, disrupts the Tsc1-Tsc2 complex and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, all Akt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking Akt phosphorylation sites. Thus, Tsc2 seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway (Potter, 2002).

Germline mutations in LKB1, TSC2, or PTEN tumor suppressor genes result in hamartomatous syndromes with shared tumor biological features. The recent observations of LKB1-mediated activation of AMP-activated protein kinase (AMPK) and AMPK inhibition of mTOR through TSC2 prompted an examination of the biochemical and biological relationship between LKB1 and mTOR regulation. LKB1 is required for repression of mTOR under low ATP conditions in cultured cells in an AMPK- and TSC2-dependent manner, and Lkb1 null MEFs and the hamartomatous gastrointestinal polyps from Lkb1 mutant mice show elevated signaling downstream of mTOR. These findings position aberrant mTOR activation at the nexus of these germline neoplastic conditions and suggest the use of mTOR inhibitors in the treatment of Peutz-Jeghers syndrome (Shaw, 2004).

Constitutive activation of the mTOR-S6K in Tsc mutants

Tuberous sclerosis (TSC) is a autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, and characterized by benign hamartoma growth. A murine model of Tsc1 disease has been developed by gene targeting. Tsc1 null embryos die at mid-gestation from a failure of liver development. Tsc1 heterozygotes develop kidney cystadenomas and liver hemangiomas at high frequency, but the incidence of kidney tumors is somewhat lower than in Tsc2 heterozygote mice. Liver hemangiomas are more common, more severe and cause higher mortality in female than in male Tsc1 heterozygotes. Tsc1 null embryo fibroblast lines have persistent phosphorylation of the p70S6K (S6K) and its substrate S6, that is sensitive to treatment with rapamycin, indicating constitutive activation of the mTOR-S6K pathway due to loss of the Tsc1 protein, hamartin. Hyperphosphorylation of S6 is also seen in kidney tumors in the heterozygote mice, suggesting that inhibition of this pathway may have benefit in control of TSC hamartomas (Kwiatkowski, 2002).

TSC2 mediates cellular energy response to control cell growth and survival

Mutations in either the TSC1 or TSC2 tumor suppressor gene are responsible for Tuberous Sclerosis Complex. The gene products of TSC1 and TSC2 form a functional complex and inhibit the phosphorylation of S6K and 4EBP1, two key regulators of translation. TSC2 is regulated by cellular energy levels and plays an essential role in the cellular energy response pathway. Under energy starvation conditions, the AMP-activated protein kinase (AMPK) phosphorylates TSC2 and enhances its activity. Phosphorylation of TSC2 by AMPK is required for translation regulation and cell size control in response to energy deprivation. Furthermore, TSC2 and its phosphorylation by AMPK protect cells from energy deprivation-induced apoptosis. These observations demonstrate a model where TSC2 functions as a key player in regulation of the common mTOR pathway of protein synthesis, cell growth, and viability in response to cellular energy levels (Inoki, 2003b).

TSC2 regulates VEGF through mTOR-dependent and -independent pathways

Inactivation of the TSC2 tumor suppressor protein causes tuberous sclerosis complex (TSC), a disease characterized by highly vascular tumors. TSC2 has multiple functions including inhibition of mTOR (mammalian target of Rapamycin). TSC2 regulates VEGF through mTOR-dependent and -independent pathways. TSC2 loss results in the accumulation of HIF-1alpha and increased expression of HIF-responsive genes including VEGF. Wild-type TSC2, but not a disease-associated TSC2 mutant, downregulates HIF. Rapamycin normalizes HIF levels in TSC2-/- cells, indicating that TSC2 regulates HIF by inhibiting mTOR. In contrast, Rapamycin only partially downregulates VEGF in this setting, implying an mTOR-independent link between TSC2 loss and VEGF. This pathway may involve chromatin remodeling since the HDAC inhibitor Trichostatin A downregulates VEGF in TSC2-/- cells (Brugarolas, 2003).

Mutation in either TSC1 or TSC2 causes the autosomal dominant disorder tuberous sclerosis, in which widespread hamartomas are seen, some of which have a high level of vascularization. Tuberous sclerosis complex (TSC) gene products negatively regulate mammalian target of rapamycin (mTOR) activity. Vascular endothelial growth factor (VEGF) is secreted by Tsc1- or Tsc2-null fibroblasts at high levels compared with wild-type cells. In Tsc1+/- mice, serum levels of VEGF are increased and appear to be associated with the extent of tumor development. Rapamycin, a mTOR inhibitor, reduces the production of VEGF by Tsc1- and Tsc2-null fibroblasts to normal levels. Moreover, short-term treatment of Tsc1+/- mice with rapamycin at 20 mg/kg leads to some changes in tumor morphology and a reduction in serum VEGF levels. These observations have three implications: (1)TSC gene products regulate VEGF production through a mTOR signaling pathway; (2) serum VEGF levels may be a useful clinical biomarker to monitor the progression of TSC-associated lesions; (3) rapamycin or related inhibitors of mTOR may have therapeutic benefit in TSC both by direct tumor cell killing and by inhibiting the development of TSC lesions through impairment of VEGF production (El-Hashemite, 2003).

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex

In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. In contrast, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. This study reports, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2 (Massey-Harroche, 2007).

Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse

Tuberous sclerosis complex (TSC) is an autosomal dominant, tumor predisposition disorder characterized by significant neurodevelopmental brain lesions, such as tubers and subependymal nodules. The neuropathology of TSC is often associated with seizures and intellectual disability. To learn about the developmental perturbations that lead to these brain lesions, a mouse model was created that selectively deletes the Tsc2 gene from radial glial progenitor cells in the developing cerebral cortex and hippocampus. These Tsc2 mutant mice were severely runted, developed post-natal megalencephaly and died between 3 and 4 weeks of age. Analysis of brain pathology demonstrated cortical and hippocampal lamination defects, hippocampal heterotopias, enlarged dysplastic neurons and glia, abnormal myelination and an astrocytosis. These histologic abnormalities were accompanied by activation of the mTORC1 pathway as assessed by increased phosphorylated S6 in brain lysates and tissue sections. Developmental analysis demonstrated that loss of Tsc2 increased the subventricular Tbr2-positive basal cell progenitor pool at the expense of early born Tbr1-positive post-mitotic neurons. These results establish the novel concept that loss of function of Tsc2 in radial glial progenitors is one initiating event in the development of TSC brain lesions as well as underscore the importance of Tsc2 in the regulation of neural progenitor pools. Given the similarities between the mouse and the human TSC lesions, this model will be useful in further understanding TSC brain pathophysiology, testing potential therapies and identifying other genetic pathways that are altered in TSC (Way, 2009).

PIP4kgamma is a substrate for mTORC1 that maintains basal mTORC1 signaling during starvation

Phosphatidylinositol-5-phosphate 4-kinases (PIP4ks) are a family of lipid kinases that specifically use phosphatidylinositol 5-monophosphate (PI-5-P) as a substrate to synthesize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Suppression of PIP4k function in Drosophila results in smaller cells and reduced target of rapamycin complex 1 (TORC1) signaling. This study shows that the gamma isoform of PIP4k stimulates signaling through mammalian TORC1 (mTORC1). Knockdown of PIP4kgamma reduced cell mass in cells in which mTORC1 is constitutively activated by Tsc2 deficiency. In Tsc2 null cells, mTORC1 activation was partially independent of amino acids or glucose and glutamine. PIP4kgamma knockdown inhibited the nutrient-independent activation of mTORC1 in Tsc2 knockdown cells and reduced basal mTORC1 signaling in wild-type cells. PIP4kgamma was phosphorylated by mTORC1 and associated with the complex. Phosphorylated PIP4kgamma was enriched in light microsomal vesicles, whereas the unphosphorylated form was enriched in heavy microsomal vesicles associated with the Golgi. Furthermore, basal mTORC1 signaling was enhanced by overexpression of unphosphorylated wild-type PIP4kgamma or a phosphorylation-defective mutant and decreased by overexpression of a phosphorylation-mimetic mutant. Together, these results demonstrate that PIP4kgamma and mTORC1 interact in a self-regulated feedback loop to maintain low and tightly regulated mTORC1 activation during starvation (Mackey, 2014).

gigas: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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