p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110β isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway regulates glucose/nutrient homeostasis and cell survival and plays a central role in both normal metabolism and cancer. The PTEN tumor suppressor gene (29, 30, 54) negatively regulates the PI3K/AKT pathway by dephosphorylating the D3 hydroxyl subunit of phosphoinositide-3,4,5-trisphosphate, a key membrane phosphatidylinositol generated by PI3K (34). PTEN undergoes genetic or epigenetic inactivation in many malignancies, including glioblastoma, melanoma, and endometrial, prostate, and breast cancers, among others (6, 13, 22, 23, 47, 49-51, 55, 68). Similarly, germ line mutations of PTEN are associated with the development of hamartomatous neoplasias such as Cowden disease and Bannayan-Zonana syndrome (17, 21, 41).The tumor suppressor function of PTEN undergoes dynamic regulation involving both C-terminal phosphorylation and protein-protein interactions. Phosphorylation of serine and threonine residues at the PTEN C-terminal tail, mediated by kinases such as CK2 and glycogen synthase kinase 3β, alters its conformational structure and association with PDZ domain-containing proteins and attenuates PTEN enzymatic activity (1, 11, 20, 32, 45, 61-63, 66, 67, 71). Conversely, PTEN function is promoted in large part through its stabilization in unphosphorylated form by incorporation into a high-molecular-weight protein complex (the PTEN-associated complex [PAC]) (66). We first demonstrated the existence of the PAC through gel filtration studies of rat liver extracts, which identified PTEN within a high-molecular-mass peak (>600 kDa), as well as a low-molecular-mass peak (40 to 100 kDa) in which PTEN is monomeric and phosphorylated (66). Subsequently, several PDZ domain-containing proteins were shown to interact with PTEN, including MAGI-1b, MAGI-2, MAGI-3, ghDLG, hMAST205, MSP58/MCRS1, NHERF1, and NHERF2, which mediate indirect binding with platelet-derived growth factor (PDGF) receptor β (25, 36, 42, 57, 66). More recently, LKB1, a serine/threonine kinase tumor suppressor (7), was also found to interact with and phosphorylate PTEN in vitro (36). In aggregate, these data suggest that PTEN functional output is controlled by a complex interplay of protein interactions and regulation of C-terminal phosphorylation.Beyond these interactions, there is also evidence to support additional regulatory mechanisms by which the tumor suppressor function of PTEN is mediated. The herpesvirus-associated ubiquitin-specific protease was shown to interact directly with PTEN and promote its nuclear entry (53). Both ubiquitination and relocalization into the nucleus constitute important PTEN regulatory mechanisms (53, 64). In many tumors, PTEN nuclear exclusion has been associated with poor cancer prognosis and more aggressive cancer development (15, 44, 56). Moreover, successful treatment of acute promyelocytic leukemia was shown to be associated with an increase in monoubiquitinylation and relocation of PTEN into the nucleus (53).Like PTEN, the p85 regulatory subunit of PI3K serves as a prominent modulator of PI3K/AKT signaling. p85, which exists in three isoforms (α, β, and γ), targets the catalytic (110-kDa) PI3K subunit to the membrane, which brings it into proximity with membrane-associated phosphatidylinositol lipids. In the steady state, p85 forms a tight association with the catalytic PI3K subunit, usually p110α or p110β in nonhematopoietic cells, with p110δ predominating in leukocytes (19). Consistent with this notion, p85 and p110 exist in equimolar ratios in a wide variety of mammalian cell lines and tissues (19), although some studies have suggested a role for free p85 in cell signaling (33, 65).Several recent lines of evidence have begun to support a possible regulatory relationship between PTEN and p85 (reviewed in references 3 and 53). For example, liver-specific deletion of PIK3R1, which encodes the p85α regulatory subunit, reduces both the activation of PI3K and PTEN enzymatic activity in this context. As a result, p85α-deficient hepatic cells express elevated levels of phosphoinositide trisphosphate and exhibit prolonged AKT activation (60). In addition, both PTEN and p85 are regulated by small GTPase proteins such as RhoA, but PTEN coimmunoprecipitates with the RhoA effector Rock only in the presence of PI3K (18, 31, 37). Although only correlative in nature, these findings may suggest a possible role for PTEN in p85 regulation or vice versa, in addition to its known function as a direct antagonist of the PI3K/AKT pathway (3, 9, 52, 57, 60).In the present study, we demonstrate an endogenous association between p85 and PTEN. Using newly generated antibodies that selectively recognize the PTEN C-terminal tail in its unphosphorylated form, we demonstrate that this PTEN-p85 association preferentially involves the unphosphorylated form of PTEN. The specificity of this interaction was confirmed using multiple antibodies and through studies of both human cancer cells and murine embryonic fibroblasts (MEFs) deficient for specific p85 subunits. This association, which also engages p110β, is enhanced by trastuzumab treatment and correlates with diminished AKT phosphorylation. These results support a functional role for the PTEN-p85 association that may have important biological and therapeutic implications for PI3K/AKT pathway regulation.     

Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P₂ through headgroup and 2′ acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P₂-containing membranes, that PI(4,5)P₂ binding tolerates 2′ acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P₂ analogues do not compete effectively with PI(4,5)P₂-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P₂-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves several functions in the viral replication cycle. One essential function is to target PrGag proteins to their assembly sites at the plasma membranes (PMs) of infected cells (4, 5, 11, 16, 25, 29, 30, 33, 35, 39, 43-45, 47, 50, 54, 56, 57). A second function is the recruitment of the viral surface/transmembrane (SU/TM; also referred to as gp120/gp41) envelope (Env) protein complex into virions (14, 15, 18, 19, 27, 51-53). In addition to these activities, numerous reports have attributed nucleic acid binding properties to retroviral MAs (24, 38, 47), and with some viruses MA appears to serve in an encapsidation capacity (24). While no encapsidation role has been assigned for HIV-1 MA, experiments have shown that MA can substitute for the HIV-1 nucleocapsid (NC) protein assembly function (38) under some circumstances, presumably by virtue of its facility to concentrate PrGag proteins by binding them to RNAs (38).A number of structural studies have been conducted on HIV-1 MA (1, 22, 41, 42, 49). The protein is N terminally myristoylated and composed of six α helices, capped by a three-strand β sheet (7, 22, 41, 42, 49). The protein trimerizes in solution and in crystals (22, 28, 49) and recently has been shown to organize as hexamers of trimers on lipid membranes (1). The membrane binding face of HIV-1 MA is basic, fostering its ability to associate with negatively charged phospholipid headgroups (1, 22, 30, 41, 42, 49). The importance of such an interaction has been underscored in molecular genetic experiments which demonstrated that depletion of PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] reduced the assembly efficiency of HIV-1 (9, 36). Consistent with these observations, HIV-1 MA preferentially binds to soluble PI(4,5)P₂ mimics through contacts with the headgroup and 2′ acyl chain, and binding promotes exposure of the MA myristate group and protein oligomerization (17, 21, 40-43, 46). However, PI(4,5)P₂ is not the only lipid to demonstrate an association with HIV-1. In particular, HIV-1 appears to assemble at cholesterol-rich PM sites, cholesterol is highly enriched in HIV-1 virions, and cholesterol depletion reduces viral infectivity (2, 6, 8, 20, 23, 26, 31, 34, 37). The HIV-1 lipidome shows additional differences from the PM lipids of infected cells (2, 5, 8), suggesting that other lipids could affect PrGag-membrane binding or virus assembly site selection.To gain a better understanding of the functions and interactions of HIV-1 MA, we have examined the liposome and nucleic acid binding properties of purified myristoylated MA. Using liposome flotation assays and a novel liposome bead binding assay, we have demonstrated that the PI(4,5)P₂ binding specificity of MA is enhanced by cholesterol, that protein myristoylation increases membrane binding efficiency but not specificity, and that 2′ acyl chain variation is compatible with PI(4,5)P₂ binding. We also examined whether soluble PI(4,5)P₂ mimics could compete with liposomes for MA binding. Surprisingly, we found that soluble mimics not only failed to compete with PI(4,5)P₂ liposomes but also increased MA binding to membranes that do not contain acidic phospholipids. Finally, we have observed that while MA does bind nucleic acids, nucleic acid binding is outcompeted by PI(4,5)P₂-containing liposomes. Our results suggest models for PrGag-membrane and RNA association and the HIV-1 assembly pathway.     

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice (for a review, see reference 42). Friend SFFV is a replication-defective virus with deletions in its env gene, giving rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity to the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice (49). The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (18, 37), as follows: the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases (11-12). Susceptibility to SFFV-induced disease is associated with expression of a short form of the receptor tyrosine kinase Stk, termed sf-Stk, that is transcribed from an internal promoter within the Stk gene of Fv-2-susceptible (Fv-2^ss) mice but not Fv-2-resistant (Fv-2^rr) mice (37) and is abundantly expressed in erythroid cells (11). Infection of erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemia-inducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation (42). Previous studies demonstrated that this Epo-independent erythroblastosis is due to the cell surface interaction of the SFFV envelope protein with the Epo receptor (EpoR) and sf-Stk (31). While interaction with the EpoR appears to be responsible mainly for the induction of Epo-independent differentiation (52), Epo-independent erythroid cell proliferation depends upon activation of sf-Stk. We recently demonstrated that sf-Stk covalently interacts with SFFV-P gp55 in hematopoietic cells that express the EpoR and that this interaction induces sf-Stk activation (31). Furthermore, exogenous expression of sf-Stk, but not a kinase-inactive mutant of sf-Stk, in bone marrow cells from sf-Stk null mice can restore Epo-independent erythroid colony formation in response to SFFV infection (5, 41). Thus, the SFFV envelope glycoprotein induces Epo-independent proliferation of erythroid cells mainly by activating sf-Stk. While sf-Stk is a key susceptibility factor for erythroblastosis induced by both SFFV-P and SFFV-A (18), it is not required for the induction of erythroblastosis by the SFFV mutant BB6, which encodes an envelope glycoprotein, gp42, that is deleted in the membrane-proximal extracellular domain (19) and does not induce sf-Stk activation (31). gp42 of SFFV-BB6 appears to exert its biological effects on erythroid cells by efficiently interacting with the EpoR (9). Compared with wild-type SFFV, SFFV-BB6 causes a relatively indolent and slowly developing disease in mice (19).A number of signaling pathways normally activated in erythroid cells after erythropoietin (Epo) binds to its cell surface receptor (40) are constitutively activated in erythroid cells infected with SFFV. These include JAK/STAT, Ras/Raf/mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, and the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathways (24, 25, 28-30, 32). SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17, 31, 43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8, 20, 21) or by SFFV through sf-Stk (5, 14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5, 29). The class IA subclass of PI3-kinase is a heterodimer comprising the p110 (α, β, δ) catalytic unit and one of five regulatory subunits (85α, p55α, p50α, 85β, and 55γ) (15). The first 3 regulatory subunits are all splice variants of the same gene (pik3r1). Deletion of pik3r1, which encodes p85α, p55α, and p50α, is lethal (6, 7), and these regulatory subunits of PI3-kinase are required for normal murine fetal erythropoiesis in mice (10).To determine the role of p85α in SFFV-induced erythroleukemia, we used a distinct nonlethal pik3r1 knockout mouse which lacks only the p85α regulatory subunit of PI3-kinase (45, 47), allowing the study of SFFV-induced erythroleukemia in adult mice. Our results indicate that p85α regulates SFFV-induced erythroid hyperplasia induced in vivo by sf-Stk-dependent, but not sf-Stk-independent, isolates of the virus as well as stress-induced erythropoiesis and suggest that this regulation may occur through the interaction of sf-Stk with p85α.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

The Silencing Domain of GW182 Interacts with PABPC1 To Promote Translational Repression and Degradation of MicroRNA Targets and Is Required for Target Release

GW182 family proteins are essential in animal cells for microRNA (miRNA)-mediated gene silencing, yet the molecular mechanism that allows GW182 to promote translational repression and mRNA decay remains largely unknown. Previous studies showed that while the GW182 N-terminal domain interacts with Argonaute proteins, translational repression and degradation of miRNA targets are promoted by a bipartite silencing domain comprising the GW182 middle and C-terminal regions. Here we show that the GW182 C-terminal region is required for GW182 to release silenced mRNPs; moreover, GW182 dissociates from miRNA targets at a step of silencing downstream of deadenylation, indicating that GW182 is required to initiate but not to maintain silencing. In addition, we show that the GW182 bipartite silencing domain competes with eukaryotic initiation factor 4G for binding to PABPC1. The GW182-PABPC1 interaction is also required for miRNA target degradation; accordingly, we observed that PABPC1 associates with components of the CCR4-NOT deadenylase complex. Finally, we show that PABPC1 overexpression suppresses the silencing of miRNA targets. We propose a model in which the GW182 silencing domain promotes translational repression, at least in part, by interfering with mRNA circularization and also recruits the deadenylase complex through the interaction with PABPC1.In multicellular eukaryotes, the regulation of gene expression by microRNAs (miRNAs) is critical for biological processes as diverse as cell differentiation and proliferation, apoptosis, metabolism, and development (4). To exert a regulatory function, miRNAs associate with Argonaute proteins to form RNA-induced silencing complexes, which repress translation and trigger the degradation of target mRNAs (4, 10, 16). The extent to which translational repression and degradation contribute to silencing depends on the specific target-miRNA combination; some targets are regulated predominantly at the translational level, whereas others can be regulated mainly at the mRNA level (3). A large-scale proteomic analysis performed in parallel with measurements of mRNA levels showed that for the vast majority of miRNA targets, silencing correlates with changes at both the protein and mRNA levels (1, 27).In animal cells, the degradation of miRNA targets is initiated by deadenylation and decapping, which are followed by the exonucleolytic decay of the mRNA body (2, 3, 9, 11, 12, 17, 19, 24, 30, 31). miRNA-dependent mRNA degradation requires a variety of proteins: an Argonaute and a GW182 protein, the CCR4-NOT deadenylase complex, the decapping enzyme DCP2, and several decapping activators including DCP1, Ge-1, HPat, EDC3, and Me31B (also known as RCK/p54) (3, 6, 9, 12, 19). Several studies previously demonstrated that miRNAs trigger deadenylation and decapping even when the mRNA target is not translated (9, 12, 19, 24, 30, 31), indicating that mRNA decay is not merely a consequence of a primary effect of miRNAs on translation but rather is an independent mechanism by which miRNAs silence gene expression.Although how miRNAs trigger mRNA degradation is well established, the mechanisms driving the inhibition of translation are unclear. Multiple mechanisms have been proposed: the displacement of eukaryotic initiation factor 4E (eIF4E) from the mRNA cap structure, interference with the function of the eIF4F complex, a block of 60S ribosomal subunit joining, or an inhibition of translation elongation (4, 10, 16). Regardless of the precise mechanism, the translational repression of miRNA targets also requires GW182 family proteins (11, 13).GW182 proteins are essential components of the miRNA pathway in animal cells, as their depletion suppresses miRNA-mediated gene silencing (reviewed in references 8 and 13). Recent studies have revealed that the silencing activity of these proteins resides predominantly in a bipartite silencing domain containing the middle and C-terminal regions (14, 22, 33). The precise molecular function of the GW182 silencing domain is not fully understood, yet it is known that the domain is not required for GW182 proteins to interact with Argonaute proteins or to localize to P bodies (3, 14, 22). Furthermore, when the silencing domains of GW182 proteins are artificially tethered to mRNAs, their expression is silenced; therefore, tethering bypasses the requirement for Argonaute proteins and miRNAs (5, 22, 33). These observations suggest that the silencing domains of GW182 proteins exhibit intrinsic silencing activity and therefore likely play a role at the effector step of silencing (13, 14, 22, 33).Here we investigate what role the Drosophila melanogaster GW182 silencing domain plays in the miRNA pathway. Overall, our results reveal that the very C-terminal region of this domain is required for the release of GW182 from silenced mRNPs. Indeed, we unexpectedly found that we could detect D. melanogaster GW182 bound to miRNA targets only in cells depleted of components of the deadenylase complex. These results suggest that GW182 dissociates from Argonaute-1 (AGO1) and miRNA targets at a step of silencing downstream of deadenylation. In contrast, GW182 mutants lacking the C-terminal region remain stably bound to miRNA targets, even in wild-type cells, indicating that this region plays a role in the dissociation of GW182 from effector complexes. We further show that the bipartite silencing domain of GW182 interacts with PABPC1 and interferes with the binding of PABPC1 to eIF4G. The interaction of GW182 with PABPC1 is also required for the degradation of miRNA targets, most likely because the interaction facilitates the recruitment of the CCR4-NOT deadenylase complex. Accordingly, overexpressing PABPC1 suppresses the silencing of miRNA targets. Our findings uncover an unexpected role for PABPC1 in the miRNA pathway.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.