An Ancient, Unified Mechanism for Metformin Growth Inhibition in C. elegans and Cancer.

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. PAPERCLIP.

G protein-coupled receptors engage the mammalian Hippo pathway through F-actin: F-Actin, assembled in response to Galpha12/13 induced RhoA-GTP, promotes dephosphorylation and activation of the YAP oncogene.

The Hippo pathway, a cascade of protein kinases that inhibits the oncogenic transcriptional coactivators YAP and TAZ, was discovered in Drosophila as a major determinant of organ size in development. Known modes of regulation involve surface proteins that mediate cell-cell contact or determine epithelial cell polarity which, in a tissue-specific manner, use intracellular complexes containing FERM domain and actin-binding proteins to modulate the kinase activities or directly sequester YAP. Unexpectedly, recent work demonstrates that GPCRs, especially those signaling through Galpha12/13 such as the protease activated receptor PAR1, cause potent YAP dephosphorylation and activation. This response requires active RhoA GTPase and increased assembly of filamentous (F-)actin. Morever, cell architectures that promote F-actin assembly per se also activate YAP by kinase-dependent and independent mechanisms. These findings unveil the ability of GPCRs to activate the YAP oncogene through a newly recognized signaling function of the actin cytoskeleton, likely to be especially important for normal and cancerous stem cells.

Protein kinases of the Hippo pathway: regulation and substrates.

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 Saccharomyces 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 orthologs 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.

The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes.

The Mst1 kinase is an important regulator of murine T cell adhesion, migration, proliferation, and apoptosis. In this study, we analyze mice lacking both Mst1 and Mst2 in hematopoietic cells. Compared with wild-type mice, these double knockout (DKO) mice exhibit a severe reduction in the number of mature T cells in the circulation and in secondary lymphoid organs (SLOs). CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes in DKO mice resemble mature T cells of wild-type mice but undergo excessive apoptosis, and their egress from the thymus is reduced by >90%. Even when placed directly in the circulation, DKO SP thymocytes failed to enter SLOs. In SP thymocytes, deficiency of Mst1 and Mst2 abolished sphingosine-1 phosphate- and CCL21-induced Mob1 phosphorylation, Rac1 and RhoA GTP charging, and subsequent cell migration. When phosphorylated by Mst1 or Mst2, Mob1 binds and activates the Rac1 guanyl nucleotide exchanger Dock8, which is abundant in the thymus. Thus, the Mst1 and Mst2 kinases control Rho GTPase activation and the migratory responses of SP thymocytes.

Phosphorylation of the U(L)31 protein of herpes simplex virus 1 by the U(S)3-encoded kinase regulates localization of the nuclear envelopment complex and egress of nucleocapsids.

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U(L)31 and U(L)34 gene products. While it is clear that the viral kinase encoded by the U(S)3 gene regulates the localization of pU(L)31/pU(L)34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU(L)31 is indispensable for the protein's normal function and contains up to six serines that are phosphorylated by the U(S)3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U(S)3 kinase, changing the serines to alanine caused the pU(L)31/pU(L)34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U(S)3 kinase-dead mutant. (iv) The replacement of the six serines of pU(L)31 with glutamic acid largely restored the smooth distribution of pU(L)34/pU(L)31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U(S)3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU(L)31 by pU(S)3 represents an important regulatory event in the virion egress pathway that can account for much of pU(S)3's role in nuclear egress. The data also suggest that the dynamics of pU(L)31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.

The U(L)31 and U(L)34 gene products of herpes simplex virus 1 are required for optimal localization of viral glycoproteins D and M to the inner nuclear membranes of infected cells.

U(L)31 and U(L)34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pU(L)31, pU(L)34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a U(L)34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the U(L)34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on U(L)34 (P < 0.0001). pU(L)34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pU(L)34/gD interaction in recruiting gD to the NM. The effects of U(L)34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pU(L)31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, U(L)31 and U(L)34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.

Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

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.

Effects of lamin A/C, lamin B1, and viral US3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus U(L)34-encoded protein to the inner nuclear membrane.

Previous results indicated that the U(L)34 protein (pU(L)34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pU(L)34 and virions requires the U(S)3-encoded kinase that phosphorylates U(L)34 and lamin A/C. Moreover, pU(L)34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pU(L)34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pU(L)34 targeting to the inner nuclear membrane (INM), the localization of pU(L)34 was determined in LmnA(-/-) and LmnB1(-/-) mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of U(S)3 kinase activity. While pU(L)34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a U(S)3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pU(L)34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pU(L)34 protein in cells infected with a U(S)3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that U(S)3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pU(L)34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins.

Emerin is hyperphosphorylated and redistributed in herpes simplex virus type 1-infected cells in a manner dependent on both UL34 and US3.

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 Cdelta (PKCdelta) 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 PKCdelta 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.

US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells.

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.