Tuberous sclerosis complex (TSC) 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. Here we report that 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. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.

Transposable elements have been routinely used for genetic manipulation in lower organisms, including generating transgenic animals and insertional mutagenesis. In contrast, the usage of transposons in mice and other vertebrate systems is still limited due to the lack of an efficient transposition system. We have tested the ability of piggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, to transpose in mammalian systems. We show that PB elements carrying multiple genes can efficiently transpose in human and mouse cell lines and also in mice. PB permits the expression of the marker genes it carried. During germline transposition, PB could excise precisely from original insertion sites and transpose into the mouse genome at diverse locations, preferably transcription units. These data provide a first and critical step toward a highly efficient transposon system for a variety of genetic manipulations including transgenesis and insertional mutagenesis in mice and other vertebrates.

A genetic screen was designed in Drosophila to interrogate its genome for mutations sufficient to cause noninvasive tumors of the eye disc to invade neighboring or distant tissues. We found that cooperation between oncogenic RasV12 expression and inactivation of any one of a number of genes affecting cell polarity leads to metastatic behavior, including basement membrane degradation, loss of E-cadherin expression, migration, invasion, and secondary tumor formation. Inactivation of these cell polarity genes cannot drive metastatic behavior alone or in combination with other tumor-initiating alterations. These findings suggest that the oncogenic background of tissues makes a distinct contribution toward metastatic development.

The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B (PKB)) regulates cell growth is unknown. Here, we report that Drosophila melanogaster Akt/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1-Tsc2 complex. We show that 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.

Nuclear movement is critical during neurogenesis and neuronal migration, which are fundamental for mammalian brain development. Although dynein, Lis1, and other cytoplasmic proteins are known for their roles in connecting microtubules to the nucleus during interkinetic nuclear migration (INM) and nucleokinesis, the factors connecting dynein/Lis1 to the nuclear envelope (NE) remain to be determined. We report here that the SUN-domain proteins SUN1 and SUN2 and the KASH-domain proteins Syne-1/Nesprin-1 and Syne-2/Nesprin-2 play critical roles in neurogenesis and neuronal migration in mice. We show that SUN1 and SUN2 redundantly form complexes with Syne-2 to mediate the centrosome-nucleus coupling during both INM and radial neuronal migration in the cerebral cortex. Syne-2 is connected to the centrosome through interactions with both dynein/dynactin and kinesin complexes. Syne-2 mutants also display severe defects in learning and memory. These results fill an important gap in our understanding of the mechanism of nuclear movement during brain development.

Prior to the pairing and recombination between homologous chromosomes during meiosis, telomeres attach to the nuclear envelope and form a transient cluster. However, the protein factors mediating meiotic telomere attachment to the nuclear envelope and the requirement of this attachment for homolog pairing and synapsis have not been determined in animals. Here we show that the inner nuclear membrane protein SUN1 specifically associates with telomeres between the leptotene and diplotene stages during meiotic prophase I. Disruption of Sun1 in mice prevents telomere attachment to the nuclear envelope, efficient homolog pairing, and synapsis formation in meiosis. Massive apoptotic events are induced in the mutant gonads, leading to the abolishment of both spermatogenesis and oogenesis. This study provides genetic evidence that SUN1-telomere interaction is essential for telomere dynamic movement and is required for efficient homologous chromosome pairing/synapsis during mammalian gametogenesis.

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Here, using a Drosophila genetic model for Ras-induced tumor progression, we show a molecular link between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/beta-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic Ras(V12)-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, as JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. Our finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression.

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