Enzymic characterization of p38s and the role of p38a in the immune response

The protein kinase activities of p38a (Mpk2) and p38b were examined by using an in vitro assay. The two Drosophila p38 MAPKs were expressed as GST fusion proteins in bacteria and purified. In vitro kinase assays demonstrate that both p38a and p38b can phosphorylate Drosophila Jun and mammalian ATF2. The Drosophila p38 isoforms can also phosphorylate myelin basic protein, which is phosphorylated more effectively by PKA. These results reveal that while Drosophila p38s can phosphorylate ATF2, Drosophila Jun also serves as a substrate but to a lesser extent. In contrast, Drosophila Basket (JNK) recognizes both substrates with the same efficiency (Z. Han, 1998).

The regulation of Drosophila p38 MAPK activities by MAPK kinases Licorne (MKK3) and MKK4 was tested. cDNAs were placed under the control of the Actin5C promoter, and different combinations of the plasmids were cotransfected into Schneider S2 cells. The FLAG epitope-tagged Drosophila p38 MAPKs were immunoprecipitated, and their kinase activity was measured in vitro with ATF2 as the substrate. The results demonstrate that Licorne is an efficient activator of both p38a and p38b. Cotransfection of MKK4 under similar conditions does not activate the Drosophila p38 MAPKs but does cause activation of D-JNK. The possible role of D-MKK4 in the activation of Drosophila p38s requires further investigation (Z. Han, 1998).

The regulation of Drosophila p38s by extracellular stimuli was also examined. Epitope-tagged p38 MAPKs were expressed in S2 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. It was found that LPS increases the kinase activity of both D-p38 isoforms. Control experiments demonstrate that the exposure of S2* cells to UV light also activates the MAPKs (Z. Han, 1998).

An anti-inflammatory drug that inhibits Drosophila p38 MAPKs also modulates insect immunity gene expression. SB203580 is a pyridinyl-imidazole compound that binds to the human p38 MAPK and inhibits the production of IL-1 and tumor necrosis factor by monocytes during inflammation. The bacterially expressed kinases were first incubated either with the solvent DMSO or with various concentrations of the drug prior to the measurement of protein kinase activity. The results demonstrate that this anti-inflammatory drug is an efficient inhibitor of both Drosophila p38 MAPK isoforms. The inhibitory effect is similar to that on mammalian p38alpha, since micromolar concentrations are sufficient to almost abolish protein kinase activity (Z. Han, 1998).

Induction of the insect immune response leads to the production of antimicrobial peptides. This inductive event can be mimicked in tissue culture. To test the hypothesis that the p38 MAPKs regulate insect immunity, S2 cells were incubated in medium that contained either solvent (DMSO) or drug (SB203580) prior to LPS induction. With LPS treatment alone, the mRNA levels of two antibacterial peptide genes, Attacin and Cecropin, were induced by more than 10-fold within 3 h. The expression of Attacin and Cecropin returns to lower levels after 6 h. In the presence of SB203580, the early phase of induction of these two genes is similar to that of the DMSO control. During the later phase, 3 to 6 h after LPS treatment, when the gene expression should be reduced, Attacin is induced to a much higher level and Cecropin induction is modestly elevated. These results suggest that the targets of the drug, presumably the p38 protein kinases, are involved in the down regulation of immunity gene expression. It is surmised that p38 is involved in the attenuation of immunity gene expression during the late phase of infection. Such negative regulation may be employed to attenuate immunity gene expression to avoid overactivation, which may be harmful to the host (Z. Han, 1998).

Overexpression of p38a blocks antimicrobial peptide gene expression. To test whether the p38 protein kinases function in vivo as attenuators of immunity gene expression, transgenic flies were generated that express the p38a cDNA under the control of the heat shock promoter. A fly strain with four copies of the transgene was established through genetic manipulation. The transgenic larvae were heat shocked at 37°C to induce the expression of p38a. After heat shock, the animals were challenged by bacterial injection. Total RNA was isolated, and immunity gene expression was examined. The parental strain shows normal induction of Attacin and Cecropin. Within 3 h, the mRNA levels are more than 10-fold higher than those in the unchallenged animals. In contrast, overexpression of p38a causes significantly reduced mRNA levels of both Cecropin and Attacin. The expression of the antifungal peptide gene Drosomycin was also examined, and it appears to be affected to a lesser extent, while the expression of another antibacterial peptide gene, Diptericin, is only modestly decreased. These results suggest that the p38 MAPK pathway is used to attenuate the induced expression of a selective group of immunity genes in vivo (Z. Han, 1998).

Mekk1 acts upstream of p38

In cultured mammalian cells, the p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses. However, there is little evidence from in vivo studies to demonstrate a role for this pathway in the stress response. A Drosophila MAPK kinase kinase (MAPKKK), Mekk1, has been identified that can activate p38 MAPK. Mekk1 is structurally similar to the mammalian MEKK4/MTK1 MAPKKK. Mekk1 kinase activity is activated in animals under conditions of high osmolarity. Drosophila mutants lacking Mekk1 are hypersensitive to environmental stresses, including elevated temperature and increased osmolarity. In these Mekk1 mutants, activation of Drosophila p38 MAPK in response to stress is poor compared with activation in wild-type animals. These results suggest that Mekk1 regulation of the p38 MAPK pathway is critical for the response to environmental stresses in Drosophila (Inoue, 2001).

Two types of cDNA, Mekk1a and Mekk1b, corresponding to distinct start sites have been isolated. The sequences of the Mekk1a and Mekk1b cDNAs contain long open reading frames that predict proteins of 1571 and 1497 amino acids, respectively. Comparison of the amino acid sequence of the Mekk1 protein with the DDBJ/EMBL/GenBank database reveals significant homology to the MAPKKK family. Among members of the MAPKKK family, Mekk1 is most similar to mouse MEKK4 and human MTK1. Mekk1 and MEKK4 share 59% amino acid identity in the kinase domain. In addition, the sequence similarity between these two proteins extends outside the N-terminal non-kinase domain as well. The N-termini of Mekk1a and MEKK4 contain a proline-rich region followed by a putative pleckstrin homology (PH) domain. However, whereas MEKK4 has a Cdc42/Rac interactive binding (CRIB)-like domain just upstream of its kinase domain, Mekk1 lacks this motif (Inoue, 2001).

Mekk1 gene is expressed throughout embryonic development. There is a high level of maternal deposition similar to MKK3/licorne and p38. In the later stages, zygotic expression is present in most tissues. Whole-mount in situ hybridization also revealed that Mekk1 mRNAs are homogenously distributed in imaginal discs and the central nervous system of late third-instar larvae (Inoue, 2001).

Mekk1 was identified as a protein that specifically interacts with Licorne, an MKK that is most similar to the mammalian p38 activators MKK3 and MKK6. Mutations in the lic gene result in embryo polarity defects that correlate with changes in two oocyte-localized determinants, Oskar and Gurken, essential for posterior and dorsal specification, respectively. Thus, the Licorne/p38 MAPK signaling pathway plays an important role in the patterning of the Drosophila egg. However, in contrast to lic mutants, females homozygous for the Mekk1 loss-of-function mutation lay morphologically normal eggs. These eggs are fertilized and develop to normal adult flies. Thus, animals mutant for Mekk1 do not show a lic-deficient phenotype. Although a specific interaction can be detected between Mekk1 and Lic in the yeast two-hybrid system, Mekk1 is unable to phosphorylate Lic proteins purified from Escherichia coli in vitro. These results suggest that Mekk1 activates Drosophila p38 in animals via a MAPKK(s) that is distinct from Lic. Consistent with this possibility, Mekk1 and p38 kinases were activated in late third-instar larvae in response to environmental stress, whereas Lic kinase activity was undetectable. Thus, it is not known which MAPKK functions between Mekk1 and p38 in this signaling pathway. Remarkably, the only additional MAPKK-like kinase in the Drosophila DNA database is MKK4, which is most similar to mammalian MKK4. MKK4 is able to activate both D-JNK and D-p38b in vitro. This raises the possibility that D-MKK4 may be a component of the Mekk1/p38 pathway required for responses to environmental stresses. Indeed, osmotic stress induces phosphorylation of a protein whose molecular weight (46 kDa) is similar to that of D-MKK4. Investigations into the functional and genetic associations between Mekk1 and other conserved components of this pathway, and the identification of p38 substrates, should allow for a more complete understanding of the Drosophila Mekk1-D-p38 pathway regulating responses to environmental stresses (Inoue, 2001).

p38b, or possibly both p38s, are phosphorylated in vivo downstream of constitutively active Thick veins

To investigate whether p38b is activated by Thick veins signaling, a preliminary biochemical characterization of p38b was carried out. Immediately after heat treatment of flies, the amount of p38b immunoprecipitated by anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected to be in the 'activation loop' region recognized by MAPKK, as is the case in mammalian p38. Thus, a test was performed to see whether an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of mammalian p38 could cross-react with p38b. This anti-p-p38 antibody detects a protein with a calculated size of 42 kDa whose amount increases immediately after heat shock. This protein is also more abundant in the flies overproducing p38b regardless of heat treatment. Therefore, it is concluded that anti-phospho-p38 can cross-react with the phosphorylated form of p38b and can be used to assay recombinant p38b phosphorylation in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38 antibodies, and a drastic increase in the level of Drosophila p38-dependent phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for mammalian p38. The correlation between the phosphorylation state and kinase activity of p38b indicates that the anti-p-p38 antibody recognizes the active form of p38b. This allowed activation of p38b by TkvCA to be examined in vivo. The amount of active p38b was found to be slightly but significantly higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in yeast, which was presumed to have the same molecular mass as p38b, is also recognized by anti-p-p38 antibody. It is therefore possible that p38b, or both D-p38's, may be activated by Tkv signaling in vivo (Adachi-Yamada, 1999).

Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH2-terminal kinase

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH2-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Identified here is the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response (Sano, 2005).

The activating transcription factor/cAMP response element-binding protein (ATF/CREB) family of proteins bears a DNA-binding domain consisting of a cluster of basic amino acids and a leucine zipper that together form the so-called b-ZIP structure. These proteins can form homodimers or heterodimers by binding via their leucine zipper motifs, after which they can bind to the cyclical AMP response element (CRE: 5'-TGACGTCA-3') via their basic region. The two major subgroups of the ATF/CREB family proteins are CREB and ATF-2. The CREB subgroup includes CREB and cAMP response element modulator (CREM), whereas the ATF-2 subgroup contains ATF-2, ATFa (recently also called ATF-7), and CRE-BPa. When the Ser-133 residue of CREB is phosphorylated by cAMP-dependent protein kinase, CREB can bind to the transcriptional coactivator CREB-binding protein (CBP), which greatly stimulates the trans-activating capacity of CREB. The trans-activating capacity of ATF-2, on the other hand, is enhanced by the phosphorylation of its Thr-69 and Thr-71 residue by stress-activated protein kinases (SAPKs) such as c-Jun NH2-terminal kinase (JNK) and p38 . SAPKs are activated by various extracellular stress such as UV, osmotic stress, and inflammatory cytokines. All three members of the ATF-2 subgroup bear the trans-activation domain in their N-terminal region: this domain consists of two subdomains, namely, the N-terminal subdomain containing the well known zinc finger motif and the C-terminal subdomain containing the SAPK phosphorylation sites. The latter subdomain has a highly flexible and disordered structure. Although the coactivator CBP binds to the protein surface of b-ZIP domain of ATF-2, the cofactor that binds to the N-terminal activation domain of ATF-2 remains unknown (Sano, 2005).

The physiological roles played by ATF-2 have been analyzed by using mutant mice. Null Atf-2 mutant mice die shortly after birth and display symptoms of severe respiratory distress and have lungs filled with meconium. In the mutant embryos, hypoxia occurs, which may lead to strong gasping respiration with the consequent aspiration of the amniotic fluid containing meconium. This is due to the impaired development of cytotrophoblast cells in the placenta that in turn is caused by decreased levels of expression of the platelet-derived growth factor receptor alpha. In addition, another Atf-2 mutant mouse, which expresses only a fragment of ATF-2, exhibits lowered postnatal viability and growth, a defect in endochondrial ossification, and reduced numbers of cerebellar Purkinje cells. However, the physiological roles played by the other ATF-2 family proteins remain unknown (Sano, 2005).

In Drosophila, three members of the mitogen-activated protein kinase (MAPK) protein family have been identified: Rolled (Erk homologue), dJNK (JNK homologue, also called Basket), and dp38a and dp38b (p38 homologue). Rolled mediates various receptor tyrosine kinase signals in the process of tracheal elaboration, cell proliferation, mesodermal patterning, R7 photoreceptor cell differentiation, and differentiation of terminal embryonic structures. In contrast, the pathway containing Hemipterous (Hep; MAPK kinase [MAPKK] homologue), dJNK, and Drosophila Jun (dJun) is involved in dorsal closure during embryo development. All mutants of this pathway exhibit the dorsal open phenotype and a decreased level of the expression of Decapentaplegic (Dpp), a secretory ligand belonging to the transforming growth factor (TGF)-β superfamily, in leading edge cells. With regard to the dp38s, they are phosphorylated by various stresses, including UV, lipopolysaccharide (LPS), and osmotic stress. The phenotype resulting from the ectopic expression of the dominant negative (DN) dp38b in the wing imaginal disc indicates that dp38b functions downstream of thickvein (Tkv), a type I receptor of the Dpp ligand, in wing morphogenesis (Sano, 2005).

To determine the in vivo function of ATF-2, the Drosophila ATF-2 homologue (dATF-2) has been identified and characterized. dATF-2 is directly phosphorylated by dp38b but not by dJNK. Moreover, genetic analyses indicated that dATF-2 acts in the dp38 signaling pathway. In addition, DNA array analysis demonstrated that dATF-2 is a major transcriptional activator of osmotic stress-inducible genes (Sano, 2005).

The amino acid sequences of the b-Zip domain and the region containing the p38/JNK phosphorylation sites of mammalian ATF-2 are well conserved in dATF-2. However, dATF-2 lacks the N-terminal zinc finger domain that is conserved in the three members of the mammalian ATF-2 family (ATF-2, CRE-BPa, and ATF-a). The N-terminal zinc finger motif and the adjacent region that contains the p38/JNK phosphorylation sites in the mammalian ATF-2 together act as the transcriptional activation domain. Therefore, the mediators that regulate the transcriptional activation of mammalian ATF-2 and dATF-2 may have different characteristics (Sano, 2005).

Extracellular stress such as UV or osmotic stress induces the dp38-induced phosphorylation of dATF-2 at Thr-59 and Thr-61 and this increases the trans-activation capacity of dATF-2. Although mammalian ATF-2 is well known to be phosphorylated not only by p38 but also by JNK, this study found that dJNK neither directly phosphorylated dATF-2 nor enhanced dATF-2-dependent transcription. Furthermore, transgenic embryos expressing DN-dATF-2 or dATF-2 dsRNA did not clearly reveal the dorsal-open phenotype that is common to the Hep, Bsk, dJun, and dFos mutants. The entire amino acid sequence of JNK1 shares 65% identity with dJNK, and the ~50-amino acid stretch within the N-terminal domain of mammalian ATF-2 that contains the phosphorylation sites is also well conserved in dATF-2 (59% identity). Therefore, it is surprising that dJNK cannot phosphorylate dATF-2, unlike what is observed for mammalian JNK and ATF-2. Furthermore, it was found that although dATF-2 is phosphorylated only by dp38, dJun is phosphorylated by both dp38 and dJun. In contrast, mammalian ATF-2 is phosphorylated by both p38 and JNK, whereas Jun is phosphorylated only by JNK. It is worth noting that ATFa is not phosphorylated by JNK. This may raise the possibility that a regulation mechanism of dATF-2 resembles to that of ATFa, and that an ancestral ATF-2/CRE-BPa gene were derived from a duplicated ATFa-like gene. The relationship between SAPKs and transcription factors in Drosophila and mammals may be useful in understanding how the stress-inducible gene expression system is established during evolution (Sano, 2005).

The GAL4-dATF-2 fusions containing the N-terminal 150 amino acids had a stronger activity than those containing the N-terminal 274 amino acids, indicating that the region between amino acids 150 and 274 has a negative effect on the activation domain of dATF-2. In the case of vertebrate ATF-2, the b-ZIP DBD suppresses the ATF-2 activation domain via intramolecular interaction. This difference may suggest that the mechanism by which the C-terminal region suppresses the activation domain is different between vertebrate ATF-2 and dATF-2. It is interesting whether the region between amino acids 150 and 274 of dATF-2 affects the stability or conformation of dATF-2 protein. Wild-type dATF-2 stimulated the luciferase expression from the CRE-containing promoter under nonstimulated condition. Because the alanine mutants of Thr-59 and Thr-61 dramatically decreased this trans-activating capacity of dATF-2, phosphorylation of these residues seems to be essential for trans-activating capacity of dATF-2. These results suggest the possibility that the Thr-59 and Thr-61 residues are phosphorylated at low levels even under nonstimulated condition. This could be due to the low levels of TNF-α or IL-1 involved in serum. Alternatively, other kinase(s) also may phosphorylate these residues, because vertebrate ATF-2 is activated by Raf-MEK-ERK pathway via phosphorylation of Thr-71 (Sano, 2005).

Using two different assay systems, this study has demonstrated at the genetic level that dATF-2 acts in the dp38 signaling pathway. First, it was shown that expression of DN-dp38b enhances the aberrant wing phenotype caused by DN-dATF-2. It has been reported previously that dp38b acts downstream of the Dpp receptor Tkv, because DN-dp38b expressed in the wing imaginal disc causes a phenotype resemble to the mutant of dpp, a Drosophila homologue of mammalian bone morphogenetic protein/TGF-β/activin superfamily. Therefore, dATF-2 may functions in the Dpp signaling pathway. This may be consistent with the finding that mammalian ATF-2 is phosphorylated by TGF-β signaling via TAK1 and p38, and it then directly binds to the Smad3/4 complex to synergistically activate transcription with Smad3/4. This study also demonstrated that DN-dp38b coexpression enhances the sensitivity of embryos expressing DN-dATF-2 to high osmolarity. Thus, dATF-2 acts in the dp38 signaling pathway, at least in wing pattern formation and the response to osmotic stress. However, no oocyte defects were observed in the transgenic flies expressing DN-dATF-2, although the dp38 MAPK pathway is known to be required during oogenesis for asymmetric egg development. Thus, dATF-2 may function only in some specific events that are regulated by the dp38 signaling pathway (Sano, 2005).

DNA array analysis indicated that ~40% of the genes that are induced by osmotic stress are also regulated by dATF-2, indicating that dATF-2 is a major inducer of osmotic stress-inducible gene expression. These genes encode cell surface and cuticle proteins, transporters, and receptors, and various endopeptidases. It is not surprising that osmotic stress may increase the production of cell surface proteins, including some receptors. In addition, the endopeptidases may be produced because high osmolarity may increase the denaturation of proteins, which must then be degraded by the cell. The dATF-2 target genes also include seven immune response genes, namely, several encoding antimicrobial peptides and one encoding a peptidoglycan recognition protein, which binds to the peptidoglycans of bacterial cell walls and triggers immune responses. LPS has been shown to increase the kinase activity of dp38. Consequently, dp38-phosphorylated dATF-2 may directly induce these immune response-related genes. However, it also has been shown that overexpression of dp38 inhibits the expression of immune response genes. This could be explained by the possibility that dp38 overexpression may inhibit the p38 signaling pathway by activating negative feedback regulatory mechanisms, such as the p38α-induced decrease of MKK6 mRNA stability in mammalian cells. In Drosophila, Gram positive bacteria and fungi predominantly induce the Toll signaling pathway to activate genes such as Drosomycin, whereas Gram negative bacteria activate the Imd pathway to activate genes such as Diptericin. DNA array analysis indicated that both Drosomycin and Diptericin are regulated by dATF-2, which suggests that dATF-2 may be a component of both the Toll and Imd pathways. Further analyses of dATF-2 will most likely enhance understanding of the molecular mechanisms involved in the Drosophila immune system (Sano, 2005).

ATF-2, acting downstream of p38, regulates fat metabolism in Drosophila

ATF-2 is a member of the ATF/CREB family of transcription factors that is activated by stress-activated protein kinases such as p38. To analyze the physiological role of Drosophila ATF-2 (dATF-2), dATF-2 knockdown flies were generated using RNA interference. Reduced dATF-2 in the fat body, the fly equivalent of the mammalian liver and adipose tissue, decreased survival under starvation conditions. This was due to smaller triglyceride reserves of dATF-2 knockdown flies than control flies. Among multiple genes that control triglyceride levels, expression of the Drosophila PEPCK (dPEPCK) gene was strikingly reduced in dATF-2 knockdown flies. PEPCK is a key enzyme for both gluconeogenesis and glyceroneogenesis, which is a pathway required for triglyceride synthesis via glycerol-3-phosphate. Although the blood sugar level in dATF-2 knockdown flies was almost same as that in control flies, the activity of glyceroneogenesis was reduced in the fat bodies of dATF-2 knockdown flies. Thus, reduced glyceroneogenesis may at least partly contribute to decreased triglyceride stores in the dATF-2 knockdown flies. Furthermore dATF-2 was shown to positively regulat dPEPCK gene transcription via several CRE half-sites in the PEPCK promoter. Thus, dATF-2 is critical for regulation of fat metabolism (Okamura, 2007).

This study has demonstrated that dATF-2 activates dPEPCK gene transcription in part via the CRE half-sites in the dPEPCK promoter. The p38 inhibitor, SB203580, suppressed the dATF-2–dependent activation of the dPEPCK promoter, suggesting that the p38 signal positively regulates dPEPCK transcription in the fat body. These results are consistent with the observation in mammals that ATF-2 directly binds to and activates the PEPCK promoter through the p38 pathway. C/EBP was also reported to control PEPCK gene transcription in liver and can form a heterodimer with ATF-2, which binds to an asymmetric sequence composed of one consensus half-site for each monomer. Therefore, ATF-2 could regulate PEPCK gene transcription together with other factors, such as C/EBP, which forms a heterodimer with ATF-2. If dp38 is required for dATF-2–dependent activation of dPEPCK transcription, dp38 would have to be constitutively activated in the fat body. In fact, p38 was reported to be constitutively active in the mammalian liver, which may be a result of metabolic oxidative stress. Retinoic acid was also shown to activate the p38 pathway leading to ATF-2–dependent activation of PEPCK gene transcription. Furthermore, dp38 mutants were shown to be more sensitive to starvation than wild-type flies. If the p38 signaling pathway is important for triglyceride stores via glyceroneogensis, this pathway may be a useful target for antidiabetic drugs, because various inhibitors for the kinases in this pathway have already been developed (Okamura, 2007).

The Ral/exocyst effector complex counters c-Jun N-terminal kinase-dependent apoptosis in Drosophila melanogaster

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).

The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors. Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture. The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).

None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis. The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).

In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).

Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).

The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).

Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of RalG20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).

The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).

Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation. The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).

Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).

A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).

The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).

Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts. The current results can explain one of the contributions of the Ral pathway to oncogenesi: cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).

The p38 pathway regulates oxidative stress tolerance by phosphorylation of mitochondrial protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However the underlying mechanisms are largely unknown. This study demonstrates that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. The importance of MK2 was shown as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, the iron-sulfur cluster scaffold protein IscU was identified as a new substrate of MK2 both in Drosophila and mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondrial iron-sulfur cluster (Tian, 2014).

p38b: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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