Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells.

We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.

Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen and cholera toxin subunit B.

Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostate-specific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8+ T-cell responses resulting in an efficient prevention of tumor growth in mice.

TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins.

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A.

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

[Incidence of severe injuries. Results of a population-based analysis]. / Inzidenz schwerer Verletzungen. Ergebnisse einer populationsbezogenen Untersuchung.

It is still unknown exactly how many persons sustain a severe injury (ISS > or =16) in Germany each year. Considering the growing restrictions and the introduction of DRGs, it was necessary to acquire data about this rather resource-intensive aspect of trauma care. The aim of this study was therefore to assess the incidence of severe trauma within a defined population. In a retrospective study all surgical emergencies within a 5-year period (1996-2000) were reviewed. Data on type, pattern, severity of injury, and mortality were extracted from the patients' records. During the study period 454 persons sustained a severe injury (ISS > or =16), 112 individuals died at the scene of the accident, and 64 during the hospital stay. The average ISS of the surviving patients was 27 (ISS 16-75). The calculated incidence of severe trauma was 25/100,000 inhabitants per year. Extrapolated, up to 40,000 persons sustain a severe injury each year in Germany. For the first time, this study has provided data on the incidence of major trauma in Germany. Based on the acquired data and a previous cost analysis, hospital treatment costs for severely injured patients amount to up to 2 billion Euros per year in Germany.

The Spir actin organizers are involved in vesicle transport processes.

The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.

Generation dependent reduction of tTA expression in double transgenic NZL-2/tTA(CMV) mice.

Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenic mice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA(CMV) transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and beta-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/tTA(CMV) mice. Luciferase activity in NZL-2/tTA(CMV)(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA(CMV)(F2) mice, and beta-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA(CMV) double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.

Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation.

We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.

Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death.

The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2.

Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells.

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.

Independent control of cell survival by Raf-1 and Bcl-2 at the mitochondria.

Bcl-2 family proteins play a critical role in the regulation of cell survival by controlling the activation of the cell death executing caspase machinery. Recent work demonstrated that they also provide a link between growth factor signaling and cell survival control. Raf-1 has been identified initially as an essential component of the mitogenic Ras-Raf-MEK-ERK cascade. However, expression of oncogenic Raf-1 also efficiently suppresses apoptotic cell death. This process requires mitochondrial translocation of Raf-1 which can be achieved either by co-expression of the anti-apoptotic protein Bcl-2 or by fusion with the transmembrane domain of the yeast outer mitochondrial membrane protein Mas 70p. It is currently unclear how mitochondrial Raf-1 prevents apoptosis. One possible mechanism involves the phosphorylation of the pro-apoptotic protein Bad resulting in the restoration of Bcl-2 function. Alternatively, the role of Bcl-2 could be limited to the mitochondrial translocation of Raf-1 and survival signaling by Raf-1 is Bcl-2 independent. To test for the mutual requirement of Raf-1 and Bcl-2 in apoptosis suppression the individual proteins were singly tested for survival activity in a genetic background which precludes the expression of the other. The results obtained in these studies demonstrate that ablation of Raf-1 or Bcl-2 expression in fibroblast cells significantly increases the sensitivity towards doxorubicin induced cell death. Reversion of the mutant phenotype could be achieved in either case by introducing a functional bcl-2 gene or a mitochondria targeted version of oncogenic Raf-1, demonstrating that each protein by itself is sufficient to confer protection. Our data thus suggest the existence of two separate pathways of survival signaling at the mitochondria controlled either by Bcl-2 or by Raf-1.

Influenza virus-induced AP-1-dependent gene expression requires activation of the JNK signaling pathway.

Influenza A virus infection of cells results in the induction of a variety of antiviral cytokines, including those that are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected cells but not in cells that do not support viral replication. Among the AP-1 factors identified to bind to their cognate DNA element during viral infections of Madin-Darby canine kidney and U937 cells are those that are regulated via phosphorylation by JNKs. Accordingly, we observed that induction of AP-1-dependent gene expression correlates with a strong activation of JNK in permissive cells, which appears to be caused by viral RNA accumulation during replication. Blockade of JNK signaling at several levels of the cascade by transient expression of dominant negative kinase mutants and inhibitory proteins resulted in inhibition of virus-induced JNK activation, reduced AP-1 activity, and impaired transactivation of the IFN-beta promoter. Virus yields from transfected and infected cells in which JNK signaling was inhibited were higher compared with the levels from control cells. Therefore, we conclude that virus-induced activation of JNK and AP-1 is part of the innate antiviral response of the cell.

Tet-system for the regulation of gene expression during embryonic development.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

Active Ras induces heterodimerization of cRaf and BRaf.

Growth factor-induced signalling leads to activation of members of the Ras family and subsequent stimulation of different Raf isoforms. Within the mechanism of Raf activation, two isoforms of Raf, cRaf and BRaf, may cooperate. We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRAF: Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH(2) terminus did not, even in the presence of active RAS: These data suggest that Ras induces the cRaf-BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRAF: Thus, Ras-induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals.

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals.

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade.

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.

Specific function of B-Raf in mediating survival of embryonic motoneurons and sensory neurons.

Embryonic sensory and motoneurons depend on neurotrophic factors for survival. Here we show that their survival requires B-Raf, which, in this function, cannot be substituted by C-Raf. Sensory and motoneurons from b-raf-deficient mice do not respond to neurotrophic factors for their survival. However, these primary neurons can be rescued by transfection of a b-raf expression plasmid. In contrast, c-raf-deficient neurons survive in response to neurotrophic factors, similarly to neurons from wild-type mice. This points to an essential and specific function of B-Raf in mediating survival of sensory and motoneurons during development.