Targets of Activity and Protein Interactions

A mitogen-activated protein kinase (MAPK) has been cloned and sequenced from a Drosophila neoplasmic l(2)mbn cell line. The cDNA sequence analysis shows that this Drosophila kinase is a homolog of mammalian p38 MAPK and the yeast HOG1 gene and thus is referred to as Dp38 (now termed Dp38a). A distinguishing feature of all MAPKs is the conserved sequence TGY in the activation domain. Dp38 is rapidly tyrosine 186-phosphorylated in response to osmotic stress, heat shock, serum starvation, and H2O2 in Drosophila l(2)mbn and Schneider cell lines. However, unlike mammalian p38 MAPK, the addition of lipopolysaccharide (LPS) does not significantly affect the phosphorylation of Dp38 in the LPS-responsive l(2)mbn cell line. Following osmotic stress, tyrosine 186-phosphorylated forms of Dp38 MAPK are detected exclusively in nuclear regions of Schneider cells. Yeast complementation studies demonstrate that the Saccharomyces cerevisiae HOG1 mutant strain JBY10 (hog1-Delta1) is functionally complemented by Dp38 cDNA in hyperosmolar medium. These findings demonstrate that similar osmotic stress-responsive signal transduction pathways are conserved in yeast, Drosophila, and mammalian cells, whereas LPS signal transduction pathways appear to be different (S. Han, 1998).

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-kappaB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. The possible involvement of the p38 MAP kinase pathway in the Drosophila immune response has been investigated. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 (Licorne) and D-MKK4, were identified. The regulation of D-p38 MAPK activity by Licorne and D-MKK4 was tested. Licorne is an efficient activator of both D-p38a and D-p38b. Cotransfection of D-MKK4 under similar conditions does not activate the D-p38 MAPKs but does cause activation of D-JNK. The regulation of D-p38 by extracellular stimuli was also studied. Epitope-tagged D-p38 MAPKs were expressed in cultured cells, which were then treated with bacterial LPS. The D-p38 MAPK activity was measured in an immune complex assay with ATF2 as the substrate. LPS increases the kinase activity of both D-p38 MAPK isoforms. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. The role of the D-p38 MAP kinases in the regulation of insect immunity was examined. The results reveal that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide (Z. Han, 1998b).

The expression patterns of the protein kinases were examined in the developing embryo to gain information about the possible functional relationship among the MAPK and MKK isoforms. The two D-p38 genes exhibit different embryonic expression patterns. The D-p38a mRNA expression is predominantly at the preblastoderm stage, indicating a high level of maternal deposition. Zygotic expression is to low to be detected during most of the embryonic development. Northern analysis, however, demonstrates the presence of low mRNA levels throughout development. At stage 16, there is a low level of staining in the posterior region, which may correspond to the developing hindgut. The preblastoderm staining indicates that D-p38a may participate in early embryonic development. The D-p38b gene is expressed throughout embryonic development. There is a high level of maternal deposition, and at later stages, zygotic expression is present in most of the tissues. At midembryogenesis, higher levels of mRNA are detected in the developing anterior and posterior midguts (Z. Han, 1998).

To examine Lic kinase activity, Lic was prepared as a glutathione S-transferase (GST)-Lic fusion protein in bacteria and purified. GST-Lic does not appreciably autophosphorylate or phosphorylate a catalytically inactive form of Xenopus p38 (GST-p38-KN), suggesting that Lic is inactive when expressed in bacteria. The kinase activity of lic was tested in yeast using influenza virus hemagglutinin-tagged Lic (HA-Lic). Expression of HA-Lic complements a pbs2 mutant expressing p38, indicating that this fusion protein is biologically functional. After exposure to osmotic shock, HA-Lic was immunoprecipitated from total cell extracts and Lic activity was determined by an in vitro kinase assay using GST-p38-KN as a substrate. Lic can phosphorylate p38-KN. not. Furthermore, osmotic shock stimulates the kinase activity of Lic in a time-dependent manner. Taken together, these results demonstrate that Lic functions as a p38 activator, suggesting that LIC is involved in a p38 MAPK pathway in Drosophila (Suzanne, 1999).

p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its role in development remains unknown. The fruit fly, Drosophila melanogaster, has two p38 genes, D-p38a (Mpk2) and D-p38b. To elucidate the developmental function of the Drosophila p38 genes, various genetic and pharmacological manipulations were used to interfere with their functions: expression of a dominant-negative form of D-p38b, expression of antisense D-p38b RNA, reduction of the D-p38 gene dosage, and treatment with the p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc causes a decapentaplegic-like phenotype and enhances the phenotype of a dpp mutant. Inhibition of D-p38b function also causes the suppression of the wing phenotype induced by constitutively active Tkv (TkvCA). Mosaic analysis reveals that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced expression of TkvCA induces an increase in the phosphorylated active form(s) of D-p38(s). These results demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to modulate Dpp signaling (Adachi-Yamada, 1999).

Anthrax lethal factor and edema factor act on conserved targets in Drosophila

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila can be used for analyzing the mechanism of toxin action. In this study, the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, were characterized in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep/c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. It was also found that LF inhibits Ras/MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors (Guichard, 2006).

Anthrax is caused by Bacillus anthracis, a Gram-positive bacterium that infects primarily herbivores and occasionally humans. B. anthracis secretes three exotoxins [lethal factor (LF), edema factor (EF), and protective antigen (PA)] that are required for its virulence. Anthrax toxins belong to the A/B subfamily of exotoxins, in which the B subunit (PA) binds to a host membrane component and promotes the entry of catalytic A subunits (LF and EF) into host cells. PA binds to the human cell-surface receptors Tumor endothelial marker 8 or Capillary morphogenesis protein 2, two related, widely expressed transmembrane proteins of unknown function. After cleavage by furin proteases, PA becomes activated and forms a heptameric prepore, which binds three molecules of EF, LF, or a combination of both, after which the complex undergoes endocytosis. A pH drop in endocytic vesicles triggers a conformational change in the PA ring, leading to translocation of EF and LF into the cytosol. LF is a zinc metalloprotease that cleaves six of the seven known human mitogen-activated protein kinase (MAPK) kinases (MAPKKs) in their N-terminal proline-rich regulatory domain, which prevents them from binding to their substrates and thereby inhibits phosphorylation and activation of downstream MAPKs. EF, the second catalytic anthrax toxin, is a Ca2+/calmodulin-dependent adenylate cyclase with a specific activity ~1,000-fold higher than that of endogenous mammalian counterparts. Because bacteria lack calmodulin, EF becomes active only after entering host eukaryotic cells, in which it causes an unregulated rise in cAMP levels (Guichard, 2006).

Both LF and EF play a central role in anthrax pathogenesis, as demonstrated by the greatly reduced infectivity of B. anthracis strains lacking either toxin. In addition, the isolated toxins can cause death (LF) or edema (EF) when coinjected with PA. The best characterized cellular response to LF is in macrophages, which undergo programmed cell death and lysis after LF exposure. There is also evidence that LF induces defects in permeability of the vascular endothelium, which, in combination with cytokines produced by dying macrophages, may contribute to the shock-like death of animals exposed to LF. The cellular basis for EF action is less well characterized than that of LF, but it has been reported that EF blocks phagocytosis in monocytes, impairs the function of dendritic cells, and inhibits antigen presentation to T cells. In addition, this toxin causes severe tissue damage and multiple organ failure followed by rapid death in mice (Guichard, 2006).

It is noted that, while there is strong evidence for LF acting at least in part by cleaving and inactivating MAPKK targets, this protease may also have other targets contributing to its lethal effects. Another important question is how LF and EF toxins cooperate to achieve optimal virulence in the host. Recent reports indicate that EF and LF can act in either opposing or synergistic fashions depending on the cellular context. In preliminary experiments, other phenotypes caused by expression of LF and EF were observed in various cell types in addition to the expected phenotypes reported in this study. This study therefore provides a starting point for analyzing potentially novel effects of LF and EF and may lead to the identification of new targets mediating cooperative effects of these two toxins (Guichard, 2006).

B. anthracis is not known to infect hosts other than mammals. Consistent with this observation, no homolog of anthrax toxin receptors tumor endothelial marker 8 and capillary morphogenesis protein 2 is encoded by the Drosophila genome, suggesting that Drosophila is not a suitable model for infection by anthrax. This is also likely to be true for many human pathogens, which have evolved to infect mammals via multiple sequential events, including host recognition, adherence, induction of virulence genes, virulence factor delivery, or evasion of host defenses. In some cases, however, it has been possible to infect Drosophila with human pathogens, such as Vibrio cholerae, Pseudomonas aeruginosa, or Staphylococcus aureus. In contrast to infection with a pathogenic organism, expression of a single virulence factor, which affects only a limited set of conserved host targets, is more likely to produce a specific and interpretable response. Because many pathogens act on specific protein targets that have been highly conserved in Drosophila, it is anticipated that Drosophila will become a widely used in vivo system for the analysis of bacterial toxins or viral virulence factors with unknown activities or unidentified targets. In addition, toxins such as LF that have multiple host target proteins may be used to simultaneously reduce or eliminate the activities of several related proteins that perform overlapping functions. Thus, pharmacogenetic strategies can complement classic loss-of-function genetics in cases where multiple genes carry out related functions (Guichard, 2006).

The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine

The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. This study analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Dual oxidase (Duox) is reduced in p38c mutant flies. This genetic analysis shows that p38c functions in a pathway with Mekk1 and Licorne (Mkk3) to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, this study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c (Chakrabarti, 2014. PubMed ID: PubMed).

licorne: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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