SUMMARY Metformin has utility in cancer prevention and treatment, though the mechanisms for these effects remain elusive. Through genetic screening in C. elegans, we uncover two metformin response elements: the nuclear pore complex (NPC) and acyl-CoA dehydrogenase family member-10 (ACAD10). We demonstrate that biguanides inhibit growth by inhibiting mitochondrial respiratory capacity, which restrains transit of the RagA-RagC GTPase heterodimer through the NPC. Nuclear exclusion renders RagC incapable of gaining the GDP-bound state necessary to stimulate mTORC1. Biguanide-induced inactivation of mTORC1 subsequently inhibits growth through transcriptional induction of ACAD10. This ancient metformin response pathway is conserved from worms to humans. Both restricted nuclear pore transit and upregulation of ACAD10 are required for biguanides to reduce viability in melanoma and pancreatic cancer cells, and to extend C. elegans lifespan. This pathway provides a unified mechanism by which metformin kills cancer cells and extends lifespan, and illuminates potential cancer targets.

Like orthologues in other members of the subfamily Alphaherpesvirinae, the U S 3 gene of herpes simplex virus type 1 (HSV-1) encodes a serine/threonine kinase (13, 37). These proteins have been implicated in nuclear egress, prevention of apoptosis, and modulation of the actin cytoskeleton to promote the cell-to-cell spread of virions (11,14,19,22,25,42,44,46,52). The current study focuses on the role of the U S 3 gene-encoded kinase activity in nuclear egress of nucleocapsids and virions.Although models of HSV virion egress differ as to the extent of its contribution, all models propose that at some point during the course of infection with wild-type herpesviruses, nucleocapsids assemble in the nucleoplasm and bud through the inner nuclear membrane (INM) and into the perinuclear space (20,51,53). This compartment is delimited by the INM and outer nuclear membrane (ONM) and is continuous with the lumen of the endoplasmic reticulum. To become enveloped, capsids must bypass the nuclear lamina, a fibrous meshwork lining the nucleoplasmic face of the INM. The nuclear lamina provides structural rigidity to the nucleus and is essential for transcription and DNA replication (15,48). The lamina contains a series of type 5 intermediate filaments composed of lamin types A, B1, B2, and C; types A and C are products of RNA splice variants from the LmnA transcript, whereas types B1 and B2 are derived from other genes (12,16,17). Like all intermediate filaments, lamins comprise globular head and tail domains that flank a rod domain (12). The rod domains of two lamins intertwine to form protomers, whereas regions bordering the rod/head and rod/tail domains likely interact with other lamin protomers to form longer filaments (40). The globular domains interact with a variety of proteins in the lamina and INM.One remarkable feature of the lamina is its dynamic nature. The lamina expands by the addition of protomers during interphase, is completely disassembled prior to mitosis, and is partially disrupted during apoptosis. Phosphorylation likely plays a role in lamin dynamics in all phases of the cell cycle. The disassembly of the lamina during mitosis is associated with phosphorylation of lamin A/C by cdc2 kinase at Ser 390 and Ser 392 and during apoptosis by protein kinase C (PKC) delta (5,7,8,32,33). Protein kinase C can phosphorylate lamin A/C at Ser 572 in vitro (8).The architecture of the nuclear lamina is altered from its normal state during HSV-1 infection (3,31,40,47,49). Depending on the cell line and time after infection, these changes include (i) limited displacement and conformational changes of lamin A/C (3, 40, 49), (ii) redistribution of lamin B to a perinuclear region (31,47), and (iii) increased mobility and mislocalization of lamin B receptor, one of several integral membrane proteins that anchor the nuclear lamina to the INM (47, 48). In attempts to understand the mechanism(s) by which * Corresponding author. Mailing address:

The U L 31 and U L 34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26,27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26,28). pU L 34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU L 31 and holds the latter in close approximation to the INM (16,19,26,31,36,37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU L 31 and pU L 34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U S 3 protein is a promiscuous kinase that phosphorylates pU L 31, pU L 34, and several other viral and cellular components (1,2,5,11,15,(21)(22)(23)25). In the absence of pU S 3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU L 31/pU L 34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12,14,27,29), and (iii) the onset of infectious virus production is delayed (21,29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U S 3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3,4,9,10,18,30,33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU S 3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU S 3 kinase responsible for the altered localization of the pU L 31/pU L 34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU L 34 was not responsible for the nuclear egress defects induced by the absence of pU S 3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU S 3-mediated phosphorylation of pU L 31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U S 3 kinase-dead phenotype, including the ...

The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP, promotes integrin clustering, actin remodeling and motility while restraining the proliferation of naïve T cells. This review will summarize current knowledge of the structure and regulation of the kinases Hippo/Mst1&2, their noncatalytic binding partners, Salvador and the Rassf polypeptides, and their major substrates Warts/Lats1&2, Trc/ndr1&2, Mats/Mob1 and FOXO.

Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase C␦ (PKC␦) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKC␦ that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.During primary envelopment, herpes simplex virus type 1 (HSV-1) nucleocapsids translocate from the nucleus to the cytoplasm by budding into the inner nuclear membrane and then fusing with the outer nuclear membrane. The capsid does not, however, have unimpeded access to the inner nuclear membrane. Lining the inside of the inner nuclear membrane is the nuclear lamina, which is composed of a meshwork of proteins with spaces too small for the capsid to move through without some disruption (2,19,65). The lamina meshwork is made up of intermediate filament family proteins called lamins that are linked to the inner nuclear membrane and to intranuclear proteins by association with lamin-associated proteins (LAPs) (reviewed in reference 65). Connection of the network of lamin proteins to the inner nuclear membrane is mediated by integral membrane LAPs, including emerin, lamin B receptor, LAP2-␤, and MAN-1 (26).Emerin is a member of a family of nuclear envelope proteins that share a common sequence called the LEM domain that mediates association with BAF (barrier to autointegration factor) and is important for the assembly of LEM domain proteins into the re-forming nuclear envelope following mitosis (21,37,39). Emerin also contains a lamin-binding domain that helps retain it in the interphase nuclear envelope (6,14,25,37). Emerin is ubiquitously expressed but is not essential for the viability of cells in culture (36). Failure to ...

In mice lacking both Mst1 and Mst2 in the lymphoid compartment, thymocyte development is normal, but single-positive thymocytes exhibit excessive apoptosis and greatly diminished thymic egress, accompanied by loss of chemokine activation of RhoA and Rac1.

Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.Most enveloped viruses assemble their capsids in the cytoplasm and then become enveloped by budding from the plasma membrane or into cytoplasmic (CT) membranes. Viruses that assemble capsids in the nucleus face the fundamental problem of transporting capsids across the nuclear envelope (NE). Nonenveloped viruses, such as polyomaviruses, that are relatively small can move through nuclear pores, and larger nonenveloped viruses, such as adenoviruses (Ad), disrupt the NE (12, 55). Herpesvirus capsids are too large to move through nuclear pores (18), and, instead, herpesviruses encode machinery which promotes envelopment at the inner nuclear membrane (NM) followed by fusion with the outer NM (29, 52). There appears to be very rapid fusion at the NE because few enveloped herpesvirus particles are normally observed within the perinuclear space. Once present in the cytoplasm, nonenveloped capsids acquire a secondary envelope, producing virions that bud into the Golgi apparatus or trans-Golgi network (13, 29, 52). These mature virions are then secreted from cells.Some aspects of the envelopment of herpesviruses at the inner NM are known. Here, the viruses encounter a major obstacle, the nuclear lamina, a rigid network of lamin proteins lining the inner NM. Herpesviruses disrupt the nuclear lamina in order to assemble along the inner surface of the NM...

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, we identify that the N-terminal domain of Dab2 interacts with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse ®broblasts attenuates canonical Wnt/b-catenin-mediated signaling, including accumulation of b-catenin, activation of b-catenin/ T-cell-speci®c factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a timedependent dissociation of endogenous Dab2±Dvl-3 and Dvl-3±axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2±Dvl-3 association and subsequent loss of Dvl-3±axin interactions. In addition, we ®nd that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo ®broblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear b-catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the b-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor. IntroductionThe Wnt family of secreted glycoproteins play key roles in embryonic development, regulating cell proliferation, motility and cell fate (Cadigan and Nusse, 1997;Dale, 1998) and in adult tissues where aberrant Wnt signaling has been shown to contribute to a variety of human cancers (Polakis, 2000;Bienz and Clevers, 2000). Wnt ligand binding to its cognate receptors can stimulate several distinct signaling pathways including the canonical Wnt±b-catenin and non-canonical planar cell polarity± convergent extension (PCP±CE) pathways. Required for activation of both of these pathways is the common mediator dishevelled (Dvl) which interprets signals from various receptors and transmits them to different effector molecules. In the canonical pathway, activation of the frizzled and LRP5/6 co-receptors results in recruitment of Dvl which relays the signal to a complex composed of adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3b (GSK3b) and b-catenin (Polakis, 2000;Wharton, 2003). In the absence of Wnt signaling, b-catenin is phosphorylated on N-terminal serine and threonine residues, targeting it for degradation by the ubiquitin±proteasome pathway (Kitagawa et al., 1999). In the presence of Wnt, association of Dvl with axin prevents GSK3b from phosphorylating b-catenin, leading to stabilization of b-catenin and its translocation to the nucleus. In the nucleus, b-catenin complexes with members of the T-cell-speci®c factor/lymphoid enhancerbinding factor 1 (TCF/LEF-1) transcription factor family to regulate target genes which include c-myc and cyclin D1 (He et al., 1998;Shtutman et al., 1999). PCP signaling, responsible for proper orientation of photoreceptor cells in Drosophila, and vertebrate CE, responsi...