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Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells. Our screen identified a group of 310 high confidence factors. These highlight the conservation of the process across eukaryotes and reveal the intricate connectivity of 60S subunit maturation with other key cellular processes, including splicing, translation, protein degradation, chromatin organization and transcription. Intriguingly, we also identified a cluster of hits comprising metabolic enzymes of the polyamine synthesis pathway. We demonstrate that polyamines, which have long been used as buffer additives to support ribosome assembly in vitro, are required for 60S maturation in living cells. Perturbation of polyamine metabolism results in early defects in 60S but not 40S subunit maturation. Collectively, our data reveal a novel function for polyamines in living cells and provide a rich source for future studies on ribosome synthesis., Nucleic Acids Research, 50 (5), ISSN:1362-4962, ISSN:0301-5610

Background: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR . The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25 , are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25 , overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

Starch forms semi-crystalline, water-insoluble granules, the size and morphology of which vary according to biological origin. These traits, together with polymer composition and structure, determine the physicochemical properties of starch. However, screening methods to identify differences in starch granule size and shape are lacking. Here, we present two approaches for high-throughput starch granule extraction and size determination using flow cytometry and automated, high-throughput light microscopy. We evaluated the practicality of both methods using starch from different species and tissues and demonstrated their effectiveness by screening for induced variation in starch extracted from over 10,000 barley lines, yielding four with heritable changes in the ratio of large A-granules to small B-granules. Analysis of Arabidopsis lines altered in starch biosynthesis further demonstrates the applicability of these approaches. Identifying variation in starch granule size and shape will enable identification of trait-controlling genes for developing crops with desired properties, and could help optimise starch processing. ISSN:0144-8617 ISSN:1879-1344

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes., Author summary Cells that capture or eat bacteria must swiftly kill them to prevent pathogens from surviving long enough to escape the bactericidal pathway and establish an infection. This is achieved by the rapid delivery of components that produce an antimicrobial environment in the phagosome, the compartment containing the captured microbe. This is essential both for the function of immune cells and for amoebae that feed on bacteria in their environment. Here we identify a central component of the pathway used by cells to deliver antimicrobial components to the phagosome and show that bacteria survive over three times as long within the host if this pathway is disabled. We show that this is of general importance for killing a wide range of pathogenic and non-pathogenic bacteria, and that it is physiologically important if cells are to avoid infection by the opportunistic human pathogen Legionella.

Objective— Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport. Approach and Results— Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of 125 I-HDL but not 125 I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells. Conclusions— The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall.

Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended “on-target” mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by “off-target” effects resulting from partial complementarity of siRNAs to multiple mRNAs via the “seed” region (i.e., nucleotides 2–8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying “seed reagents” for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.

The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3 + endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3 + virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.

X-ray analysis of polycrystalline powder samples has grown beyond its roots in the world of laboratory research and is regarded as one of the most powerful industrial process-control tools in the field of building materials and minerals. This is the key to characterize the element and the phase composition of the material. This is largely due to the development of industrial X-ray analytical systems, which have transformed these advanced analytical techniques born in the laboratory into a robust, workmanlike and easy-to-use tool for today’s heavy industries. X-ray diffraction (phase analysis) opens enormous possibilities for process and quality control. Moreover, the recent development of ultra-high-speed X-ray detectors allows for “on the fly” quantitative X-ray diffraction analysis and truly interactive process control. Hydration of cements can be studied relative ease. Additionally Computed X-ray Tomography (CT) can yield valuable information in the study of mortars and concrete.

Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

SummaryDuring natural transmission, bunyaviruses are introduced into the skin through arthropod bites, and dermal dendritic cells (DCs) are the first to encounter incoming viruses. DC-SIGN is a C-type lectin highly expressed on the surface of dermal DCs. We found that several arthropod-borne phleboviruses (Bunyaviridae), including Rift Valley fever and Uukuniemi viruses, exploit DC-SIGN to infect DCs and other DC-SIGN-expressing cells. DC-SIGN binds the virus directly via interactions with high-mannose N-glycans on the viral glycoproteins and is required for virus internalization and infection. In live cells, virus-induced clustering of cell surface DC-SIGN could be visualized. An endocytosis-defective mutant of DC-SIGN was unable to mediate virus uptake, indicating that DC-SIGN is an authentic receptor required for both attachment and endocytosis. After internalization, viruses separated from DC-SIGN and underwent trafficking to late endosomes. Our study provides real-time visualization of virus-receptor interactions on the cell surface and establishes DC-SIGN as a phlebovirus entry receptor.

Summary The Bunyaviridae constitute a large family of enveloped animal viruses, many members of which cause serious diseases. However, early bunyavirus-host cell interactions and entry mechanisms remain largely uncharacterized. Investigating Uukuniemi virus, a bunyavirus of the genus Phlebovirus, we found that virus attachment to the cell surface was specific but inefficient, with 25% of bound viruses being endocytosed within 10 min, mainly via noncoated vesicles. The viruses entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomes. Acid-activated penetration, occurring 20–40 min after internalization, required maturation of early to late endosomes. The pH threshold for viral membrane fusion was 5.4, and entry was sensitive to temperatures below 25°C. Together, our results indicate that Uukuniemi virus penetrates host cells by acid-activated membrane fusion from late endosomal compartments. This study also highlights the importance of the degradative branch of the endocytic pathway in facilitating entry of late-penetrating viruses., Graphical Abstract Highlights ► Endocytosis of the bunyavirus Uukuniemi is mainly clathrin independent ► After uptake, the virus particles are routed via early to late endosomes ► Virus penetration occurs by acid-activated membrane fusion from late endosomes

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 A resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

Protein kinase B (PKB) is a member of the second messenger subfamily of protein kinases. The three isoforms of PKB identified have an amino-terminal pleckstrin homology domain, a central kinase domain, and a carboxy-terminal regulatory domain. PKB is the major downstream target of receptor tyrosine kinases that signal via the phosphoinositide (PI) 3-kinase. The crucial role of lipid second messengers in PKB activation has been dissected through the use of the PI 3-kinase-specific inhibitors wortmannin and LY294002. Receptor-activated PI 3-kinase synthesises the lipid second messenger PI-3,4,5-trisphosphate, leading to the recruitment of PKB to the membrane. Membrane attachment of PKB is mediated by its pleckstrin homology domain binding to PI-3,4,5-trisphosphate or PI-3,4-bisphosphate with high affinity. Activation of PKB α and β is then achieved at the plasma membrane by phosphorylation of Thr308/309 in the A-loop of the kinase domain and Ser473/474 in the carboxy-terminal regulatory region, respectively. The upstream kinase that phosphorylates PKB on Thr308, termed PI-dependent protein kinase-1, has been identified and extensively characterised. A candidate for the Ser473/474 kinase, termed the integrin-linked kinase, has been identified recently. Activated PKB is implicated in glucose metabolism, transcriptional control, and in the regulation of apoptosis in many different cell types. Stimulation of PKB activity protects cells from apoptosis by phosphorylation and inactivation of the pro-apoptotic protein BAD. These results could explain why PKB is overexpressed in some ovarian, breast, and pancreatic carcinomas.

To study the role of protein kinase B (PKB) in response to cellular stress, we examined PKBalpha activity following different stress treatments. Hyperosmotic but not chemical stress resulted in inactivation of PKBalpha and prevented activation by pervanadate and mitogens. Hyperosmotic shock did not affect the MAP kinase pathway, suggesting that this inhibitory effect was specific for PKB. Our data further indicate that downregulation occurs via dephosphorylation of Thr308 and Ser473, the major regulatory phosphorylation sites of PKBalpha. Indeed, calyculin A, which inhibits protein phosphatases 1 and 2A, effectively blocked hyperosmotic stress-mediated inactivation (dephosphorylation) of PKBalpha. High osmolarity did not affect phosphatidylinositol 3-kinase activity but led to a marked increase in PI(3,4,5)P3 and a decrease in PI(3,4)P2 formation after pervanadate stimulation, suggesting that hyperosmotic stress has an inhibitory effect on a phosphatidylinositol 5-phosphatase which converts PI(3,4,5)P3 into PI(3,4)P2. Immunofluorescence studies revealed that membrane translocation, a prerequisite for PKB activation, was not affected by hyperosmotic stress. Our results indicate that hyperosmotic stress can act at two levels: (i) inhibition of phosphorylation of Thr308 and Ser473 by upstream kinases and (ii) by promoting rapid dephosphorylation of these regulatory sites.

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus.

Proceedings of sessions of GeoShanghai 2010, held in Shanghai, China, June 3-5, 2010. Hosted by Tongji University, China; Shanghai Society of Civil Engineering, China; Chinese Institution of Soil Mechanics and Geotechnical Engineering, China. In cooperation with Alaska University Transportation Center, USA; Geo-Institute of ASCE, USA; Deep Foundation Institute, USA; East China Architectural Design and Research Institute Company, China; Georgia Institute of Technology, USA; Nagoya Institute of Technology, Japan; Transportation Research Board, USA; University of Newcastle, Australia; University of Illinois at Urbana-Champaign, USA; University of Kansas, USA; University of Tennessee, USA; Vienna University of Natural Resources and Applied Life Sciences, Austria. This Geotechnical Special Publication contains 41 papers addressing many different areas of soil behavior, constitutive modeling, and geo-micromechanics. Papers cover topics ranging from experimental studies of soil shear strength and compressibility to theoretical advances in Biot’s consolidation theory and constitutive modeling. The research described in this proceedings advances understanding of soil as an engineering material and improves our ability to model the behavior of soil in slopes, foundations, and earth structures.

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.

Protein kinase B (PKB) is a member of the second messenger-dependent family of serine/threonine kinases that has been implicated in signaling pathways downstream of growth factor receptor tyrosine kinases and phosphatidylinositol 3-kinase. Here we report the characterization of the human beta-isoform of PKB (PKBbeta). PKBbeta is ubiquitously expressed in a number of human tissues, with mRNA and protein levels elevated in heart, liver, skeletal muscle, and kidney. After transfection into HEK-293 or COS-1 cells, PKBbeta is activated 2- to 12-fold by mitogens and survival factors. Activation was due to phosphorylation on Thr-309 and Ser-474, which correspond to Thr-308 and Ser-473 implicated in the regulation of PKBalpha. Both phosphorylation and activation were prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Moreover, membrane-targeted PKBbeta was constitutively activated when overexpressed in HEK-293 cells. Although the specific activity of PKBbeta was lower than that of PKBalpha toward Crosstide as a substrate (23 nmol/min/mg compared with 178 nmol/min/mg for PKBalpha), both enzymes showed similar substrate specificities. Using confocal microscopy, we show that activation of PKBbeta results in its nuclear translocation within 20 to 30 min after stimulation. These observations provide evidence that PKBbeta undergoes nuclear translocation upon mitogenic activation and support a role for PKB in signaling from receptor tyrosine kinases to the nucleus through phosphatidylinositol 3-kinase.

Lamins constitute the nuclear lamina, which underlie the inner membrane of the cell nucleus. Phosphorylation of lamins is a key factor in the regulation of nuclear structure during the cell cycle and of gene transcription. Since an uncontrolled cell cycle and altered gene transcription are major characteristics of neoplasms, we looked for differences in lamin B2 phosphorylation between PBMC, ALL and AML cells. Using different lamin B2-specific antibodies, we detected two different lamin B2 species termed lamin B2 and B2A. Although phosphorylation of lamin B2 in leukemic cells was reminiscent of resting cells, the majority of ALL and AML samples showed significantly higher and more altered lamin B2A phosphorylation compared to PBMC. It remains to be elucidated which mechanism leads to these alterations and whether it could explain the extended G1-phase frequently observed in ALL cells.

The phosphorylation of signal transduction molecules is of central importance for growth factor-induced transduction of mitogenic signals, cell growth and differentiation. Stimulation of growth factor-receptor uses by their ligands can activate several signalling modules depending on the cell type. Activation of receptor tyrosine tease cascades can be either independent of a second messenger system or dependent on a second messenger.

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6.

The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739–746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 μM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.

International audience; Numerical modeling of the behavior of geotechnical structures subjected to cyclic loading requires the consideration of two main aspects: the constitutive relationship and numerical strategy. The constitutive model has to be representative of the clay behavior, whereas the numerical strategy should be efficient in order to reduce the computation time. To simulate undrained triaxial cyclic tests on clay, a method of time homogenization is applied to a bounding surface plasticity model. This method of homogenization is based upon splitting time into two separate scales. The first scale relates to the period of cyclic loading and the second to the characteristic time of the material. Simulations of undrained triaxial cyclic tests on normally consolidated clay under one-way cyclic loading are carried out. The performance of time homogenization is numerically validated. The sensitivity of the time increment is also investigated