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

Gene name - gigas

Synonyms - TSC2

Cytological map position -

Function - signal transduction protein

Keywords - cell cycle, TOR pathway, tumor suppressor

Symbol - gig

FlyBase ID: FBgn0005198

Genetic map position - 3-46

Classification - Rap1GAP domain

Cellular location - probably cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Recent literature
Housden, B. E., Valvezan, A. J., Kelley, C., Sopko, R., Hu, Y., Roesel, C., Lin, S., Buckner, M., Tao, R., Yilmazel, B., Mohr, S. E., Manning, B. D. and Perrimon, N. (2015). Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal 8: rs9. PubMed ID: 26350902
Summary:
The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. This study developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, synthetic interactions with TSC1 and TSC2 were identified. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder affecting 1 in 5800 individuals. TSC occurs in multiple organs, including the brain, eyes, skin, kidney, heart, lungs, and skeleton, and is characterized by the presence of benign tumor cells termed hamartomas. Hamartomas are a mass of disorganized but differentiated cells indigenous to the site. TSC hamartomas rarely progress to malignancy, but brain hamartomas frequently cause epilepsy, mental retardation, autism, or attention deficit-hyperactive disorder. One of the notable features of TSC hamartomas is the presence of giant cells in the tumors. Linkage studies in families with TSC have established two TSC loci, TSC1 (see Drosophila Tsc1) and TSC2, each accounting for approximately 50% of cases. The TSC1 gene encodes a novel protein, hamartin, that contains a single transmembrane domain and a large cytoplasmic tail with coiled-coil domains (van Slegtenhorst, 1997). The TSC2 gene, the homolog of Drosophila Gigas and the subject of this overview, encodes a novel protein, tuberin, that contains a region of homology to the GTPase-activating protein (GAP) for the small-molecular-weight GTPase Rap1. Clones of gigas mutant cells induced in imaginal discs differentiate normally to produce adult structures. However, the cells in these clones are enlarged (gigas means 'giant' in Latin) and repeat S phase without entering M phase. This result suggests that the TSC disorder may result from an underlying defect in cell cycle control (Ito, 1999 and references). Recent studies have focused on the role of the TSC genes as modifiers of the insulin pathway (Tapon, 2001, Potter, 2001 and Gao, 2001). In particular, it is unlikely that deficiencies in TSC gene function results in alterations in cell polyploidy (Ito, 1999), but instead the growth and size defects may arise through defective signaling in the insulin pathway (Tapon, 2001, Potter, 2001 and Gao, 2001).

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb, a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin (TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2/Gigas functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).

Mutations in the TSC1 and TSC2 genes have been described in patients with tuberous sclerosis (Wilson, 1996 and van Slegtenhorst, 1997). Moreover, loss of heterozygosity at the TSC1 and TSC2 loci has been demonstrated in TSC patient lesions as well as in sporadic tumors of non-TSC patients (Green, 1994; Henske, 1995, and van Slegtenhorst, 1997). These results support a tumor suppressor function for both TSC1 and TSC2. The mechanism by which the loss of hamartin or tuberin produces tumors is unknown. The clinical features of TSC1 and TSC2 disease are indistinguishable (Povey, 1994), suggesting that the two TSC proteins participate in the same biochemical process. Since TSC mutations seem to affect cell growth, TSC proteins might be involved in the regulation of the cell cycle (Ito, 1999 and references).

The FRT/FLP recombination system (Xu, 1993) was used to screen the left arm of the third chromosome for mutations affecting eye development. About 150,000 X-ray-mutagenized progeny were examined for abnormal morphology in eye clones and 20 complementation groups of lethal mutations were isolated. One of these groups (C1), comprised of eight alleles, contains mutations in gigas, a previously described mutant that exhibits a very similar large cell phenotype (Ferrús, 1976 and Canal, 1994). gigas was originally isolated as a mutant with larger bristles in clones (Ferrús, 1976), and gigas mutant photoreceptors in eye clones are two to three times larger and establish more synapses than normal neurons (Canal, 1994). Homozygous gigas animals are larval lethal and die by early third instar. C1 mutations produce enlarged cells in mutant clones in the eye and wing. All unit eyes (ommatidia) in mutant clones are two to three times larger in area than normal. Eye sections reveal that all the cells, including photoreceptor cells and nonneuronal accessory cells, are enlarged in the clone; however, the structure and organization of ommatidia are nearly normal. Photoreceptors are occasionally missing, especially at clone borders where there are both normal and enlarged cells in the same ommatidia. Although C1 mutant clones consist of larger cells, the developmental program of these cells seems to proceed normally. When mutant clones are generated in the wing, sensory bristles in the clones are larger but appear otherwise normal. In C1 clones on the wing blade, hair density is decreased. Since all wing blade cells have a single hair at the same position as each another, this result indicates that individual epidermal cells are larger. These results demonstrate that multiple cell types are affected by C1 (Ito, 1999).

Mutations have been characterized in both Tsc1 and Tsc2/gigas genes of Drosophila. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The disease phenotype in humans and observations in Drosophila suggest that all the functions of TSC1 and TSC2 are mediated by a complex of the two proteins, and that neither protein has additional functions. Moreover, combined overexpression of both Tsc1 and Tsc2 readily elicits a phenotype in a variety of tissues, while overexpression of either protein alone is insufficient. This argues that most, if not all, of the endogenous Tsc1 and Tsc2 is sequestered in the complex and is unavailable to complex with exogenously supplied protein. Cells mutant for either Tsc1 or Tsc2 are larger than wild-type cells and show a marginal decrease in their division time. Thus, the rate of mass accumulation (growth) in Tsc1 and Tsc2 mutant cells must be greater than that of wild-type cells to allow them to be larger than their neighbors despite a similar division time. Hence, the primary alteration in the mutants may be an increase in the rate of cellular growth. Conversely, when both Tsc1 and Tsc2 are overexpressed, the growth rate of the tissue is reduced. The cells are smaller and take longer to divide than wild-type cells. The increase in duration of the cell cycle may represent an attempt to maintain a near normal cell size in a situation where the rate of growth is reduced. An alternate possibility is that Tsc1 and Tsc2 negatively regulate both cell growth and cell cycle progression independently (Tapon, 2001).

The increased growth rate of mutant tissue is apparent in both cycling and postmitotic cells. In cycling populations such as the wing disc and in the eye disc anterior to the morphogenetic furrow, mutant clones are larger than the corresponding wild-type twin spots. The difference in size between the clone and the twin-spot becomes more exaggerated after the cells have stopped dividing, resulting in a greatly increased representation of mutant tissue in the adult (Tapon, 2001).

A major pathway that regulates growth in Drosophila is the pathway that links signaling via the insulin receptor to phosphorylation of the ribosomal protein S6. This is thought to lead to increased ribosome biosynthesis and mass accumulation. Thus, loss-of-function mutations in the insulin receptor (inr), the insulin receptor substrate (chico), and in genes encoding the downstream signaling molecules PI3K, Akt (Dakt), and S6 kinase (dS6K), all reduce cell and organ size. Conversely, overexpression of PI3K or loss-of-function mutations in the insulin pathway antagonist PTEN (dPTEN) lead to increased cell and tissue growth. In addition, Ras1, possibly acting via dmyc, can also promote cell growth in response to extracellular growth factors. Increased activity of Cyclin D in a complex with cdk4 has also been shown to be a potent stimulus for growth in both dividing and postmitotic cells (Tapon, 2001 and references therein).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

Imaginal discs containing large mutant clones of either Tsc1 or Tsc2 are significantly larger than wild-type imaginal discs but are patterned relatively normally. Mutations in several cell size regulators such as E2F or Rbf have been shown to affect the size of individual cells but do not alter the final size of the organ. The mechanisms that define organ size are poorly understood. The complex of Tsc1 and Tsc2 restricts the growth of organs in vivo. Screens for genes that interact with Tsc1 and Tsc2 are likely to identify additional regulators of organ size (Tapon, 2001).

Tsc1 and Tsc2 may modulate the cell cycle via changes in cyclin levels. In Tsc1 and Tsc2 mutant clones, the levels of both Cyclin E and Cyclin A are elevated. Cell growth driven by dmyc or Target of rapamycin (dTor) elevates Cyclin E levels. It has been postulated that Cyclin E may function as a 'growth sensor' in a manner analogous to CLN3 in yeast and that the translation of Cyclin E is more efficient in cells that have an increased rate of growth. The increased levels of Cyclin E may be responsible for the shortening of G1 in Tsc1 and Tsc2 mutants. It is unclear why Cyclin A and Cyclin B are also elevated in mutant cells. Cyclin A is normally expressed at high levels in G2. In Tsc1 or Tsc2 mutants, Cyclin A levels are elevated in the post-mitotic cells of the eye disc that are clearly not arrested in G2. Thus it seems likely that the increased growth in mutant cells may also lead to increased levels of mitotic cyclins. Alternatively Tsc1 and Tsc2 may function in a pathway that negatively regulates cyclin levels (Tapon, 2001).

While the increased levels of cyclins are likely to be a response to the increased growth rate of mutant cells, the possibility that they are in some way responsible for the increased growth rate cannot be excluded. In Drosophila, the Cyclin D/cdk4 complex serves to promote growth. In such a scenario, the loss of Tsc1 or Tsc2 gene function may lead to elevated levels of cyclins leading to increased growth and proliferation. Surprisingly, increased expression of Cyclin E, which is thought to primarily promote S-phase entry and not growth, is also able to suppress the phenotype induced by overexpression of Tsc1 and Tsc2. This might reflect the existence of feedback loops where Cyclin E might downregulate the levels or activity of the Tsc1/Tsc2 complex. Alternatively, in some circumstances, Cyclin E might assume some of the functions of the growth promoting Cyclin D. Indeed, in mammalian cells, cyclin E has been shown to fully compensate for the loss of cyclin D1 (Tapon, 2001).

Tsc mutant cells fail to maintain a developmentally induced G1 arrest posterior to the second mitotic wave in third instar eye imaginal discs. The establishment of this G1 arrest requires a downregulation of Cyclin E and Cyclin A expression. However, the transient cell cycle arrest in the morphogenetic furrow occurs normally in Tsc1 and Tsc2, suggesting that it is the maintenance of G1 arrest that is perturbed rather than its initial establishment. Postmitotic cells continue to grow abnormally in Tsc1 and Tsc2 mutants and express elevated levels of Cyclin E and Cyclin A. A likely model is that inappropriate and continued growth in postmitotic cells leads to an accumulation of Cyclin E and the mitotic cyclins. This would eventually force cells to overcome a developmentally regulated cell cycle arrest and to reenter the cell cycle. Indeed, many of the lesions in patients with TSC occur in organs that consist predominantly of postmitotic cells such as the heart and brain. A successful therapeutic strategy in tuberous sclerosis is likely to be one that can curtail the inappropriate cell growth (Tapon, 2001).


GENE STRUCTURE

Transcript length - 6.2 kb

Genomic length - 22 kb

Exons - 16


PROTEIN STRUCTURE

Amino Acids - 1847

Structural Domains

No yeast or C. elegans homologs of either TSC1 or TSC2 were detected. Sequence alignment with the human and the mouse TSC2 proteins shows a high degree of conservation. The human TSC2 protein is 26% identical (46% similar) to the Gigas protein. The highest level of similarity (53% identity) is found in the 164 amino acids of the putative Rap1GAP domain (RGAP). The presence of conserved arginine fingers in GAP proteins are important for their catalytic activity. TSC2/Gigas proteins have putative arginine fingers that do not resemble those of other known GAP subfamilies. Thus, TSC2 proteins might constitute a new subfamily of GAP proteins. Human TSC2 proteins were shown to have GAP activity in vitro for Rap1 (Wienecke, 1995) and Rab5 (Xiao, 1997), although the significance of these activities in vivo remains to be evaluated (Ito, 1999).


gigas: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 June 2001

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.