InteractiveFly: GeneBrief

p38a MAP kinase: Biological Overview | References

BIOLOGICAL OVERVIEW

The p38 mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signalling mechanism involved in processes as diverse as apoptosis, cell fate determination, immune function and stress response. Aberrant p38 signalling has been implicated in many human diseases, including heart disease, cancer, arthritis and neurodegenerative diseases. To further understand the role of p38 in these processes, a Drosophila strain was generated that is null for the D-p38a gene. Mutants are homozygous viable and show no observable developmental defects. However, flies lacking D-p38a are susceptible to some environmental stresses, including heat shock, oxidative stress and starvation. These phenotypes only partially overlap those caused by mutations in D-MEKK1 and dTAK1, suggesting that the D-p38a gene is required to mediate some, but not all, of the functions ascribed to p38 signalling (Craig, 2004).

p38/HOG1 was first identified in yeast as a gene required for osmotic maintenance. p38 signalling has since been shown to be important in organisms as diverse as plants and humans, and in processes including apoptosis, cell fate determination, immunity and stress responses. Aberrant p38 signalling has been implicated in many human diseases, including heart disease, cancer, arthritis and neurodegenerative disease, making it an attractive target for pharmaceutical intervention. For this reason, defining the processes regulated by p38 in vivo is essential (Craig, 2004).

The mitogen-activated protein kinase (MAPK) signalling cascade consists of three components: MAPK, MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). The main families of MAPKs are the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs. Like all MAPKs, p38s are serine/threonine kinases, which contain a canonical TGY dual phosphorylation motif (Craig, 2004).

Four vertebrate p38 genes have been identified: α, β, δ and γ. However, the requirements for p38 in development and homeostasis remain the least clearly defined of the MAPKs. Murine p38α knockout mice are embryonic lethal and show defects in angiogenesis. Single gene knockouts for MKK3 and MKK6 are viable and lack developmental defects, whereas the phenotype of double MKK3 and MKK6 knockouts mimics the p38α mutant, making detailed in vivo analysis difficult. Mutations that disrupt p38 pathway signalling in Caenorhabditis elegans result in mild defects in olfactory neuron cell fate determination, sensitivity to high metal ion concentrations and starvation, or pathogen resistance (Craig, 2004).

Two Drosophila p38 MAPK genes, D-p38a and D-p38b, have been identified. D-p38 activity is increased in Drosophila cell lines in response to a variety of environmental stimuli, including osmotic shock, heat shock, oxidative stress, immune stimulation, serum starvation and UV radiation (S. J. Han, 1998; Z. S. Han, 1998). The MAPKKs D-MKK3/licorne and D-MKK4 have been shown to activate both p38 isoforms in vitro (S. J. Han, 1998; Z. S. Han, 1998). A small deletion that removes several genes including D-MKK3/licorne results in embryos with axis determination defects, although interpretation of these data is complicated by the presence of other genes in the deletion. Additionally, the Drosophila genome contains two MAPKKK genes implicated in p38 signalling. Both D-MEKK1 and dTAK1 (TGF-Activated Kinase) mutants have been isolated, and are homozygous viable and fertile, with no observed developmental defects. D-MEKK1 mutants show stress response phenotypes including sensitivity to heat shock and osmotic stress, but have a normal immune response, whereas dTAK1 mutants have a strong immune response deficiency phenotype. Overexpression screens indicate that TGF-β/dTAK1 signalling during development may act through JNK, D-p38a or D-p38b, depending on the overexpression system and tissue used (Adachi-Yamada, 1999; Takatsu, 2000; Mihaly, 2001). The relationship between D-MEKK1 and the p38 kinases is complex (Inoue, 2001); phosphorylation of p38 in response to heat shock and osmotic stress is reduced but not abolished in D-MEKK1 null mutants, indicating that another MAPKKK is also partially responsible for p38 phosphorylation in the stress response (Craig, 2004).

Importantly, no mutations in either Drosophila p38 gene have been reported so far. This paper reportd the phenotypes of a complete deletion of D-p38a. Mutants show only partial phenotypic overlap with D-MEKK1, sharing susceptibility to some environmental stresses but not to others. D-p38a mutants show no detectable developmental defects, suggesting that D-p38a and D-p38b may be partially redundant as mediators of at least some aspects of MAPKK/MAPKKK activity (Craig, 2004).

To address the role of p38 signalling in vivo, a D-p38a null strain, D-p38a¹, was generated using a series of P-element-mediated mutagenesis strategies. The EP(3)3637 P-element (820 base pairs (bp) upstream of the D-p38a locus was mobilized. A new line was recovered that contained a second P-element insertion, EP(3)3637-2, 263 bp downstream of the D-p38a locus. These two elements were then simultaneously excised, creating a complete deletion of the D-p38a locus. The original EP(3)3637 was precisely excised, leaving both flanking genes intact, as determined by direct sequencing. Reverse transcription-PCR (RT-PCR) confirmed that this strain is null for the D-p38a locus (Craig, 2004).

Unlike mouse p38α mutants, which are embryonic lethal (Adams, 2000; Allen, 2000; Tamura, 2000), D-p38a¹ flies are viable and fertile, and show no developmental abnormalities. No gross defects in patterning or apoptosis were observed in developing embryonic or larval tissues, including the embryonic nervous system, or the leg, wing and eye imaginal discs, in contrast to mutants of the Drosophila EGFR/Ras/MAPK and JNK pathways. Expected mendelian ratios of viable progeny were observed from crosses of mutants to flies heterozygous for a chromosomal deficiency of the D-p38a region (48% D-p38a¹/+ versus 52% D-p38a¹/deficiency, n=353). In addition, no interaction was observed when one allele of jnk/bsk¹ or jnk/bsk² was introduced into a D-p38a¹ background; deficiencies removing one copy of D-p38b or both MKK3/lic and MKK7/hep also had no effect. Reducing D-p38b activity by overexpression of a D-p38b antisense construct (Adachi-Yamada, 1999) in a wild-type or D-p38a¹ background similarly resulted in no developmental defects. Loss of one copy of D-p38a suppressed the ommatidial polarity phenotype induced by overexpression of the planar polarity gene disheveled (dsh) (Paricio, 1999); however, removal of both copies of D-p38a¹ failed to modify the hypomorphic phenotype of dsh¹, making the significance of this observation unclear (Craig, 2004).

On the basis of previous work demonstrating the role of p38 signalling in environmental stress responses, the susceptibility of D-p38a mutants to a variety of such stresses was tested. Adult D-p38a¹ mutants showed reduced resistance to dry starvation. In addition, D-p38a¹ mutant flies are susceptible to 37°C heat shock. This susceptibility seems to be a specific defect in the heat-shock response, as the average lifespan of D-p38a¹ flies was unchanged compared with wild type at both 25°C and 29°C. This finding is in contrast to the phenotype observed for adult D-MEKK1 mutant flies, which were largely wild type in their response to heat shock. Lastly, D-p38a¹ mutants are susceptible to oxidative stress; flies placed on medium containing 1% H₂O₂ showed a reduced lifespan compared with both wild-type and D-MEKK1 mutant flies (Craig, 2004).

Curiously, unlike D-MEKK1 mutant flies, D-p38a¹ mutants were not susceptible to osmotic shock. The viability of D-p38a¹ homozygous and heterozygous flies was equivalent when reared on food containing 0.2 M NaCl, whereas the viability of D-MEKK1 homozygous flies was reduced, as previously reported. A smaller reduction was observed in D-MEKK1^ur36 viability than previously reported (Inoue, 2001); the line has been subsequently outcrossed and this disparity in sensitivity to osmotic shock may reflect a difference in the genetic background. Although dTAK1 mutants are immune response deficient, and overexpression of p38a in vivo impaired the innate immune response of larvae (Z. S. Han, 1998), no immune response deficiency was observed in D-p38a¹ mutant adults. Northern analysis showed that following septic injury, the inducible expression of five representative antimicrobial peptide genes, diptericin, attacinA, cecropinA, defensin and drosomycin, was similar in control and mutant flies. Moreover, survival of D-p38a mutants following bacterial infection was not compromised. Additionally, wound healing appeared normal in adults and embryos. Finally, Affymetrix oligonucleotide arrays were used to provide a more global measure of gene regulation after infection. Microarray analysis indicated that antimicrobial peptide gene expression was comparable to controls, as was the expression of other genes important in immune and stress responses: although some variation in expression levels was observed between control and mutant flies, such variation is normal for immune response genes (Craig, 2004).

One potential exception is induction of attacinA, which was consistently reduced on microarrays for D-p38a flies. However, subsequent attempts to confirm this result using RT-PCR and Northern analysis did not clearly confirm this result. There are four attacin genes (attacinA-D) in the Drosophila genome, and cross-hybridization of the attacinA probe to these other genes makes it difficult to determine which (if any) of them is defective in the response to septic injury. Because the viability of the D-p38a flies after septic injury is normal, any defect in the induction of the single attacinA gene may not have a significant effect on the overall immune response of the mutants (Craig, 2004).

To demonstrate that the stress responses are indeed due to loss of D-p38a function, rescue experiments were performed by expressing D-p38a on an inducible ubiquitous heat-shock promoter. Ectopic expression of D-p38a from a single copy of the transgene in a wild-type background did not change survival of 37°C heat shock. However, similar expression of D-p38a in a D-p38a¹ mutant background was successful in rescuing mutant survival to 86.8% of wild-type levels (Craig, 2004).

Surprisingly, a further increase in expression derived from two copies of the transgene in a D-p38a mutant background resulted in a less robust rescue of heat stress, to 75.4% of wild-type levels. In addition, expressing D-p38a with two copies of the transgene in a wild-type background further reduced the survival rate to just 33.6% of control. This suggests that levels of D-p38a above a certain threshold can inhibit the ability of flies to adapt to heat shock. Therefore, although these experiments confirm that signalling through D-p38a mediates heat-shock response, it is also clear that tight regulation of D-p38a is required for appropriate stress response signalling (Craig, 2004).

Thus, genotypically null D-p38a¹ strain lacks defects in development or apoptosis. D-p38a mutants are sensitive to some environmental stresses, such as heat shock, dry starvation and H₂O₂ exposure. D-p38a flies are not sensitive to high osmolarity or infection, distinguishing them from D-MEKK1 and dTAK1 flies, respectively. Another MAPK, such as D-p38b or JNK, may mediate these processes. Significantly, reduction of D-p38b or JNK did not show any additional phenotypes in a D-p38a null background, although the ability of residual D-p38b or JNK to provide full function cannot be ruled out. Conversely, D-p38a mutants are more susceptible to acute heat shock and oxidative stress, whereas D-MEKK1 mutants responded in a manner similar to wild type. This difference suggests that at least for some responses, D-p38a activity is independent of D-MEKK1. These phenotypes highlight the complexity of signalling in this pathway; phosphorylation of p38 in response to heat and osmotic stress is reduced but not abolished in D-MEKK1 mutants, indicating that another MAPKKK may be partially responsible for phosphorylating the p38 MAPKs (Inoue, 2001). To assess the p38 pathway function more completely, mutations in the D-MKK3/licorne, D-MKK4 and D-p38b loci will be required (Craig, 2004).

p38 signalling is a potential target for pharmacological intervention in many inflammatory diseases, including pulmonary disease, Crohn's disease and Alzheimer's disease. Clinical trials are presently underway to study the efficacy of p38 inhibitors in rheumatoid arthritis and asthma, among others. Given the chronic nature of these diseases, understanding the consequences of long-term loss of p38 signalling in vivo may be important in predicting potential negative outcomes in patients. Indeed, in one example, clinical development was abandoned after adverse neurological effects were seen in animal models, although it is unclear whether these effects were specific to the loss of p38 function or another unintended target. This study demonstrates that in vivo loss of D-p38a in Drosophila does not have any developmental consequences or affect longevity, but does confer sensitivity to specific environmental stresses (Craig, 2004).

Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-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. This study identified 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 were also observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogates 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).

MK2-dependent p38b signalling protects Drosophila hindgut enterocytes against JNK-induced apoptosis under chronic stress

The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. This study used the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, it was demonstrates that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress (Seisenbacher, 2011).

The p38 SAPK belongs to the MAPK family and is conserved from yeast to humans. In higher eukaryotes, p38 associates with its major target, the MAPK-activated protein kinase MK2. This complex resides in the nucleus in the resting state. Upon stress, p38 is activated by MKK3/MKK6 and phosphorylates MK2, which results in an exposure of the nuclear export signal of MK2 and a subsequent nuclear export of the complex (ter Haar, 2008). Another consequence of the p38/MK2 complex formation is the stabilisation of p38 protein. Interestingly, the kinase activity of MK2 is required neither for the nucleo-cytoplasmic shuttling nor for the p38 protein stabilisation (see ter Haar, 2008). Conversely, MK2 kinase activity is crucial to phosphorylate small heat-shock proteins, transcription factors (e.g., SRF and HSF-1), and Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. The inhibitory phosphorylation of TTP by the p38/MK2 complex has been shown to increase the translation of AU-rich elements (ARE)-containing mRNAs including the mRNA encoding the proinflammatory cytokine TNFα. Furthermore, p38 and MK2 have been shown to act as cytoplasmic checkpoint kinases in parallel to CHK1. Due to the plethora of p38/MK2 functions, targeting the p38 SAPK branch with inhibitors might lead to harmful side effects. Thus, it is important to understand the roles of p38 signalling in a tissue-specific context. The availability of mouse models has helped to decipher some in vivo roles of p38 SAPK signalling but the complex nature of the intestinal system has hampered a detailed analysis (Seisenbacher, 2011 and references therein).

Gut homeostasis, under normal and stress conditions, is ensured by complex interactions between the intestinal epithelium, the immune system and the gut flora. Drosophila has been used as a simple model organism to address different aspects of intestinal homeostasis. Replenishment of the gut epithelium by intestinal stem cells (ISCs) clearly contributes to epithelial homeostasis but how the differentiated enterocytes (ECs) are protected against stressors has remained largely unknown. This study used the larval hindgut of Drosophila as a simple intestinal model organ to address how stress signalling in the hindgut ECs ensures intestinal homeostasis in the absence of proliferative cells. This analysis identifies the p38b/MK2 signalling module as a critical component in the protection of hindgut ECs against salt stress (Seisenbacher, 2011).

A model is proposed that puts a p38b/MK2 complex in the centre of stress-protection of the hindgut ECs . In the absence of this signalling module, cells are undergoing JNK-dependent apoptosis upon stress. The lesion in the EC monolayer results in the damage of the overlying hindgut musculature. This regional loss of the barrier function leads to systemic defects in the larvae, further weakening the larvae and impairing growth under stress conditions. As a consequence, pathogens and toxic substances might enter the body cavity, eventually resulting in the melanisation of pericardial cells and the induction of cecropin in the midgut (Seisenbacher, 2011).

Interestingly, JNK activation in MK2 mutant hindguts precedes the melanisation, and it consistently occurs in patches. Within these areas, some cells acquire highest amounts of JNK activity and eventually undergo apoptosis. The surrounding cells maintain JNK activity, forming a rim around the scar in the tissue. The number of affected ECs remains roughly constant for a given stress. Presently it is not known what determines the patches with high JNK activity within the tissue. Although the dorsal domain (hd, positive for engrailed expression) ECs of the hindgut form a homogeneous epithelium and are facing the same stressor, JNK signalling is only induced in clusters of a certain size but not in surrounding cells. Increased JNK activation was also observed in the p38α deleted intestinal epithelium of a mouse model for inflammatory bowel diseases (IBDs). Furthermore, ulcerations occur in similar patchy patterns in IBDs. Thus, the MK2 mutant phenotype may be useful to decipher how a group of cells within a tissue of genetically identical cells transforms into the weakest link in the chain upon stressful conditions (Seisenbacher, 2011).

Several lines of evidence support the notion that the p38b/MK2 signalling complex is key to EC protection against chronic salt stress. (1) p38b and MK2 mutant larvae both develop BDs upon stress conditions. (2) The severity of the p38b; MK2 double mutant phenotype upon stress is similar to the p38b single mutant phenotype, suggesting that they act in the same signalling pathway. (3) p38b but not p38a physically associates with MK2 via its C-terminal CD domain (DPTD motif). (4) The binding of p38b to MK2 stabilises MK2. (5) Upon co-expression, p38b but not p38a redirects MK2 to the cytoplasm. (6) Both the activation and the catalytic activity of p38b are required to efficiently relocalise MK2. (7) The binding of MK2 to p38 and the catalytic activities of both kinases are essential to protect the ECs of the larval hindgut upon salt stress. Taken together, stabilisation, localisation and activation of MK2 by p38b are required for a proper stress response (Seisenbacher, 2011).

The genetic analysis also revealed that p38 SAPK signalling is contributing to stress protection in different ways in addition to the pivotal role of the p38b/MK2 complex. First, p38b impacts on hindgut homeostasis in an MK2-independent manner. This is apparent from the p38b mutant larvae that, in contrast to MK2 mutant larvae, develop BDs even at normal conditions. Consistently, a p38b protein version that no longer binds MK2 partially rescues the p38b mutant phenotype. Second, p38a is also required for full stress protection. The strong phenotype of MK2; p38a double mutants underscores the importance of the p38a SAPK pathway upon severe salt stress. However, the double mutants do not display an increase in BD formation but rather a decrease in viability. Thus, p38a may be involved in general stress protection that is not specific to the hindgut tissue. Third, a common p38 SAPK branch, encompassing p38a, p38b and potentially also p38c, is essential as the p38a; p38b double mutants die. Since the p38a¹ allele affects the coding sequences of p38a and p38c, the p38a¹; p38b^d27 double mutants may even represent p38a; p38b; p38c triple mutants. However, it is unclear to date whether p38c is a pseudogene. Recent studies have suggested an involvement of p38c function in immune gene regulation, early larval survival, and fertility. Thus, further studies will be required to clarify whether p38c contributes to p38 signalling subbranches. Finally, the slight increase in BDs seen in MK2; p38b double mutants under normal conditions suggests that MK2 also performs a p38b-independent function in the hindgut. p38b mutants always impact on MK2 signalling since the MK2 protein is not stable and probably not correctly localised if not bound to p38b. Negative feedback regulation acting from MK2 on the activation of p38b further complicates the SAPK signalling network (Seisenbacher, 2011).

What are the upstream components regulating the p38b/MK2 complex? Surprisingly, MKK3/Lic does not appear to play a role in the hindgut function of the p38b/MK2 branch. MKK3 but not MKK4 can activate p38 proteins in cell culture. On the other hand, siRNA mediated knockdown of both MKK4 and MKK3 is required to fully block the activation of p38 under certain stresses in S2 cells. Both p38b and MKK3/lic mutants show a strong reduction in p38 activation but no BDs are observed in MKK3/lic mutants. In contrast, mutants for MEKK1, which acts upstream of MKK3, do develop BDs similar to p38b and MK2 mutants. In mammalian cells, it has been shown that p38 can be activated independently of a MAP2 kinase. However, no activation of p38 occurred in fibroblasts of MKK3 MKK6 double mutant mice. Since MKK4 is a suppressor of the BD phenotype and MKK7 most likely does not activate p38 in Drosophila, a scenario of MAP2K-independent p38 activation could also apply for the p38b/MK2 signalling branch in the larval hindgut (Seisenbacher, 2011).

Interestingly, overexpression of the kinase-dead or of the non-activatable p38b protein version results in an even stronger phenotype than the deletion of p38b, probably by titrating upstream partners that would have additional functions besides activating p38b. A common p38a/p38b activator would be a strong candidate for such an upstream component. Saturating this common p38 activator with p38b^KR or p38b^AGF would essentially result in a p38a/p38b double mutant situation and therefore would explain the strong phenotype, especially at high salt conditions (Seisenbacher, 2011).

This analysis of the p38b/MK2 signalling module in hindgut ECs reveals how deletion of SAPK members results in increased sensitivity towards a particular stressor from the molecular level to the level of the whole organism. These findings provide a new model of how hindgut homeostasis is maintained and how different SAPK branches act together in vivo to ensure cellular survival upon stress. The p38 SAPK pathway efficiently protects hindgut ECs over a wide range of stress conditions and for an astonishingly long time period of at least five days without cell replenishment. In this light, a comparative analysis of the larval and the adult hindgut (which has to be maintained for a time period of up to forty days) will be of great interest (Seisenbacher, 2011).

How does p38 interfere with JNK activation in the hindgut epithelium? In mammalian cells, p38α binds to and phosphorylates TAB1 (and potentially TAB2). As a consequence, the activities of TAK1 and thereby JNK are reduced. TAB1 is not conserved in Drosophila but TAB2 might be involved in a similar negative feedback loop. TAB2 has been implicated in JNK activation in response to peptidoglycans and lipopolysaccharides. However, in the absence of TAB2, no change in JNK activation in response to Sorbitol or NaCl has been observed. Alternatively, p38 might induce a JNK phosphatase. p38α has been shown to impact on JNK activation by inducing DUSP1/MKP-1 in mammalian cells. Sustained JNK activation due to a loss of DUSP1/MKP-1 resulted in increased cell death in response to UV stress. Interestingly, p38 activation was also increased in DUSP1/MKP-1 mutant cells but p38 activity was not linked to cell death induction. Similarly, MK2 mutant larvae bearing BDs display an overactivation of both p38 and JNK, and blocking JNK only is sufficient to prevent the BD phenotype (Seisenbacher, 2011).

Despite the more complex nature of mammalian guts, the strong conservation of stress-signalling pathways and the similar demands of ECs make it likely that the current results will also be important in the context of various diseases of the intestinal system. A variety of different signalling pathways have been implicated in IBDs, underscoring the complex nature of these diseases. p38 and MK2 are critical regulators of TNFα production and are thereby associated with IBDs but the role of p38 SAPK in IBDs has remained controversial. This study identifies a crucial role of p38b/MK2 signalling in the first line of defence against a particular stressor in a model system devoid of an adaptive immune system. The consequences of lacking this immune system-independent protective function of a SAPK branch might parallel early steps of IBD development in intestinal epithelial cells. Indeed, a recent study has revealed tissue-specific effects of p38α in a DSS-induced mouse model for IBDs. Deleting p38α in the myeloid lineage had beneficial effects, consistent with the inflammatory nature of IBDs. In contrast, p38α deletion in the intestinal epithelium resulted in a loss of gut homeostasis, marked by increased proliferation and by a reduction in goblet cells. Thus, these studies on Drosophila p38 signalling and its role in the larval hindgut provide a basis to specifically address the role of ECs in the maintenance of an intestinal epithelium in the absence of proliferation and immune response (Seisenbacher, 2011).

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).

Intertissue control of the nucleolus via a myokine-dependent longevity pathway

Recent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines) responsible for this intertissue communication. This study shows that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK), whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging (Demontis, 2014. PubMed ID: 24882005).

Select septate junction proteins direct ROS-mediated paracrine regulation of Drosophila cardiac function

Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. This study has identified a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. This study has found that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. It was further determined that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. These results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology (Lim, 2019).

Cell-cell interaction is typically maintained and regulated by various multi-protein complexes such as tight junctions, adherens junctions (AJs), and gap junctions. Invertebrate septate junctions (SJs), which have functional and molecular similarity to vertebrate tight junctions (TJs), are specialized, multi-protein junctional complexes that reside between the apposed plasma membranes of adjacent epithelial cells. In Drosophila, more than 20 molecular constituents of the SJ have been identified, and characterization of these proteins reveals their canonical role in in sealing neighboring cells and restricting the free diffusion of solutes between adjacent cells, thereby providing a paracellular permeability barrier. The SJ protein complex is also involved in the coordinated changes in cell shape and rearrangement during tissue morphogenesis at a stage when the SJ structure has not yet formed or matured to become optically visible. For instance, mutations in all tested SJ genes cause defects in head involution, dorsal closure, and salivary gland elongation during early embryonic development before a clear SJ structure has been formed. Mutations in all tested SJ genes also cause cell-cell dissociation in the Drosophila embryonic heart, a tissue that seemingly lacks discernable SJs. Although most studies on the SJ proteins are focused on their canonical barrier function, it has been known that subsets of SJ proteins may have a different, non-barrier role. For instance, the SJ proteins Neurexin-IV (Nrx-IV), the Na+K+ATPase β subunit Nervana 2 (Nrv2), Coracle (Cora), and Yurt form a group with a distinct role in promoting epithelial apical-basal polarity. SJ components have also recently been found to play a role in regulating Hippo signaling to control intestinal stem cell activity and hematopoiesis. Together, these findings support the emerging notion that SJ proteins could serve important roles beyond their canonical barrier function. However, the non-barrier functions of the SJ proteins and the individual SJ proteins that could be involved remain poorly understood (Lim, 2019).

The heart is a heterogeneous organ comprising the contractile cardiomyocytes (CMs) and non-myocytes, such as the epicardial cells and endocardial cells. The non-myocytes have important signaling roles that contribute to CM development, growth, and function. The Drosophila heart is a linear tube comprising two inner rows of contractile CMs closely flanked by two outer rows of non-myocytic pericardial cells (PCs). PCs have been characterized as nephrocytes that are analogous to the mammalian podocytes that function to filter toxins and proteins from the hemolymph, the equivalent of mammalian blood. The PC nephrocytes are characterized by an intricate cell shape that includes elongated infoldings of the plasma membrane to form foot processes and labyrinthine channels. The labyrinthine channels are sealed by the slit diaphragm, which is a highly organized structure composed of similar proteins as the slit diaphragm in mammals. The slit diaphragm serves as a filtration barrier to control the inflow of certain substances into the labyrinthine channels from the hemolymph. In addition, vesicular invaginations of the plasma membrane occur along the labyrinthine channels that are indicative of endocytosis of the sequestered materials from the hemolymph. Materials endocytosed into the nephrocytes, presumably toxic molecules from the hemolymph, are targeted for either degradation in the lysosome or recycling back to the hemolymph. Moreover, the CMs and PCs are separated by a basement membrane composed of extracellular matrix (ECM), which could serve as a filtration system for hemolymph content (Lim, 2019).

On the other hand, accumulating evidence is indicating an important secretory function of PC nephrocytes. An early observation of an increased synthesis of the bactericidal enzyme lysozyme in PCs following the experimental infection of the insect Calliphora erythrocephala with bacteria provided the first indication that PCs could manufacture proteins for release into the hemolymph. More recently, Drosophila PCs have been reported to secrete factors, such as the ECM components and hemolymph proteins that could directly control neighboring CM function. In addition, PCs have been reported to produce reactive oxygen species (ROS) under normal, non-stressed conditions. ROS belong to a group of reactive chemical species produced by the incomplete reduction of molecular oxygen and are now recognized to serve an important role in the regulation of various cardiac physiological processes. Physiological ROS produced in the PCs of the Drosophila heart control the production of downstream signals such as D-p38 MAPK in PCs that then act in a paracrine manner to regulate CM function and morphology. The phenomenon is apparently conserved, as a study on the zebrafish heart reported that injury-induced H2O2 in the epicardial cells promotes the regeneration of the neighboring myocardium through the activation of ERK1/2 MAPK signaling and likely the generation of soluble factors from the epicardial cells. Together, these findings support the notion that a conserved ROS-MAPK signaling axis operates in the epi- or pericardium to influence myocardial function. However, the molecular mechanisms underlying ROS-MAPK-mediated paracrine interactions are currently unknown (Lim, 2019).

This study found that among the SJ proteins tested in adult PCs, only Cora and Kune-kune (Kune) are altered in abundance by ROS-D-p38 signaling in PCs. The results further showed that pericardial ROS-D-p38 signaling regulates CM function and structure through Cora and Kune. It was also found that Cora controls Kune amount in PCs and that pericardial Kune in turn modulates myocardial Kune expression that is essential for normal cardiac physiology. This study thereby unravels an unexpected function of the select SJ proteins Cora and Kune as physiological signaling mediators in PCs, a role that is distinct from their common primary barrier function (Lim, 2019).

On the basis of the results of this study, a model is proposed for the ROS-mediated paracrine regulation of cardiac physiology. In PCs, physiological ROS-p38 level governs Cora amount, which in turn regulates the level of Kune in the cellular surface. Peripheral Kune then directs the abundance of Kune in the CMs, which is essential for proper myocardial function and morphology. As a result, lowering of ROS-p38 signaling to sub-physiological level in PCs reduces pericardial Cora level and heightens pericardial Kune level, thereby raising Kune in CMs to a level that is detrimental to normal cardiac function. Conversely, elevating ROS-p38 signaling to supra-physiological level in PCs increases pericardial Cora quantity and diminishes pericardial Kune content, thereby suppressing Kune in the CMs to a level that perturbs normal heart function (Lim, 2019).

The findings suggest that Cora and/or Kune serve dual roles as structural elements of the SJ complex and as downstream effectors of ROS signaling. Such a dual function of Cora or Kune is unexpected but perhaps not unprecedented. The signaling role of Cora and Kune as core SJ components appears analogous to that of Arm as a core AJ component. Within the AJ, Arm mediates cell-cell adhesion and anchoring of the actin cytoskeleton. However, upon activation by Wingless, the Drosophila homolog of Wnts, Arm accumulates in the cell and serves as a key effector of Wingless signal transduction. In the case of Cora and Kune in the PCs, in response to the ROS signal, p38 is activated which then regulates the abundance and/or activity of these two individual SJ proteins. It is therefore proposed that Cora and/or Kune in the SJ have parallel functions as Arm in the AJ in that they serve as a structural component of the junctional complex and as downstream effector of signaling pathways (Lim, 2019).

The results indicate that Kune level in the PC affects Kune level in the CM; however, the underlying mechanism is unclear. One possibility is that pericardial Kune and cardiomyocyte Kune homotypically interact. In this scenario, one would predict that Kune is likely localized at the cell-cell interface. This was not observed; however, it does not necessarily rule out the homotypic interaction hypothesis. It is possible that in addition to engaging in homotypic interaction to mediate ROS signaling, other obligations of Kune may cause Kune to become more evenly distributed across the cells. For instance, Kune might be involved in the nephrocytic activity of PCs, and hence localization of Kune all over the cell surface is essential to promote the uptake of materials from the hemolymph into PCs. In the myocardium, Kune might be involved in the synchronous contraction of the CMs, a process that could be facilitated by the uniform localization of Kune across the entire CM surface. Alternatively, Kune interaction between the pericardial and cardiac cells might not involve their direct homotypic interaction but rather be mediated by the basement membrane that resides between PCs and CMs, at least in certain regions of the fly heart. In addition, an aberrant change in the pericardial nephrocyte morphology caused by loss of pericardial Kune might also alter Kune level in the CM. Last but not least, paracrine factors could be released from the PC in a Kune-controlled manner, which then influence Kune level in the CM. Regardless of whether intercellular Kune interaction occurs via direct cell-cell contact or indirect mechanisms, the results have demonstrated an interesting phenomenon by which the maintenance of normal Kune abundance in CMs by its pericardial counterpart is essential for proper adult cardiac morphology and physiology. This further raises the question as to how Kune acts in CMs to control proper cardiac performance and morphology. One possibility is that Kune regulates ion channel level and/or activity in the CM plasma membrane, such as the transient receptor potential (TRP) family of Ca2+ channels. As such, alterations in the CM Kune level could perturb intracellular Ca2+ homeostasis, thereby disrupting proper cardiac contractility and structure. These possibilities remain to be investigated in future studies (Lim, 2019).

In summary, these findings reveal that select SJ proteins can act as signaling effectors and suggest that the SJ, like the AJ, could serve to organize signaling centers. This work also provides important insights into the essential mechanisms of ROS-mediated non-myocyte-myocyte signaling interactions, a process that appears to be conserved between invertebrates and vertebrates (Lim, 2019).

Dopamine receptor Dop1R2 stabilizes appetitive olfactory memory through the Raf/MAPK pathway in Drosophila

In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. This study addresses this question in the context of sugar-rewarded olfactory long-term memory (LTM). Associative conditioning increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf or MAPK expression in KCs selectively impairs LTM but not short-term memory. Moreover, this study shows that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory (Sun, 2020).

This study supports the idea that Dop1R2 signaling through the Raf/MAPK pathway in KCs is critical in stabilizing appetitive memory. This could be achieved through acquisition or consolidation of appetitive LTM. How is post-training Dop1R2 signaling triggered in this context? Accumulating evidence implies that Dop1R2 detects the basal dopamine release after learning. In aversive olfactory learning, the post-training enhancement of the oscillatory activity of MB-projecting DANs (MB-MP1 and MB-MV1) underlies LTM consolidation, and Dop1R2 in KCs is responsible for detecting the enhanced dopamine signals. This signaling is also reported to mediate forgetting early labile memory, suggesting distinct neural mechanisms to regulate memories with different temporal dynamics. In appetitive learning, Dop1R2 is suggested to be the mediator of the oscillating DANs, which represent the energy value of the reward and consolidate LTM. Collectively, after conditioning Dop1R2 signaling upon specific reinforcement input is a conserved mechanism to stabilize LTM. As MB-projecting DANs are also engaged in conveying reward information during memory acquisition, the Dop1R2/Raf/MAPK pathway might additionally be involved during the acquisition of LTM (Sun, 2020).

In contrast to the well characterized receptor tyrosine kinase signaling, it is rather unexpected to find the Raf/MAPK pathway as a downstream target of Dop1R2, a G-protein-coupled receptor. Dop1R2 was recently shown to have a preferential affinity to the Gαq subunit to elicit a robust intracellular Ca2+ increase upon ligand stimulation in KCs. There are multiple lines of biochemical evidence suggesting that Gαq-dependent Ca2+ signals could trigger several pathways, such as small GTPase Rap1, protein kinase C, or Ras, to activate Raf. Furthermore, some reports suggested that calcium influx through N-methyl-d-aspartate receptor induces transient MAPK phosphorylation. Hence, intracellular Ca2+ might be the key second-messenger system to link Dop1R2 and Raf/MAPK in appetitive LTM (Sun, 2020).

This study found that MAPK has a pivotal role to stabilize appetitive memory in KCs. MAPK signaling is known to regulate different cellular processes ranging from cytoskeletal dynamics to transcriptional modulation. In Drosophila, a recent work unveiled that MAPK stabilizes presynaptic structural changes in KCs upon associative training with electric shocks, reportedly by changing the activity of an actin cytoskeleton regulator (Zhang, 2018). Such MAPK-induced cytoskeletal change might also occur in appetitive learning. Alternatively, a recent study showed that LTM consolidation involves MAPK translocation to the nuclei in KCs (Li, 2016). Consistently, it is reported that MAPK activates transcription factors like c-Fos and cAMP response element-binding protein (CREB) in KCs to form aversive LTM. Appetitive LTM is also dependent on CREB in KCs. Collectively, it is proposed that MAPK stabilizes appetitive memory by regulating these transcription factors. Future investigation on the downstream of the MAPK pathway should reveal the newly transcribed genes for memory stabilization (Sun, 2020).

Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. This study revealed that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. These findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. This study also exemplifies a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism (Nishida, 2021).

This study elucidated the mechanism by which Src drives cell proliferation and cell death in an obligatory coupled manner. The obligation is mediated by coupling of two MAPK pathways diverging from the lynchpin protein Slpr. Downstream of Slpr, JNK activates cell death signaling, while p38 activates cell proliferation in a methionine-Tor dependent manner. Src can potentially regulate Tor signaling through both p38-dependent and -independent mechanisms. This work provides several new insights discussed below (Nishida, 2021).

First, the findings that Slpr mediates Src signaling provide a new molecular insight into regulation of Src signaling. Drosophila Src has been known to regulate various signaling pathways, including Notch, MAPKs, Jak-Stat, EGF, Wnt, and Hippo signaling, but Slpr has not previously been implicated in Src signaling. Especially, the mechanism behind Src-mediated JNK activation was elusive in spite of its biological importance in various contexts. Slpr fills in the gap between Src and JNK. In hindsight, it may seem sensible that Slpr, a JNKKK, could link Src and JNK. However, previous studies proposed that ubiquitin E2 complex Bendless and F-actin cytoskeleton mediate Src-JNK signaling. Thus, it was unclear until now whether a MAPKKK is necessary for Src-mediated activation of JNK. Furthermore, there are five Drosophila JNKKKs, including dTAK1, Mekk1, Ask1, Wnd, and Slpr, each of which functions uniquely in a context-dependent manner. In an initial RNAi screening that identified Slpr as a Src effector, other MAPKKKs were not identified. Thus, identification of Slpr as a linker between Src and JNK provides a new insight. An urging, next question is how Src regulates Slpr. It is speculated that the components that are considered as Src downstream and/or Slpr; upstream, such as Dok, Shark, and Misshapen, may mediate the signal transduction between them. Interestingly, it was also found that Slpr inhibition suppresses the phenotype of CA Ras overexpression, which, similar to Src, simultaneously induces apoptosis and proliferation. This suggests that Slpr could function as a lynchpin hub that integrates inputs from multiple oncogenes (Nishida, 2021).

This study exclusively focused on cell autonomous signaling induced by Src. But it was noticed that Src elicits non-cell autonomous activation of MAPKs, cell death, and proliferation, This is reminiscent of the non-cell autonomous activation of Yorkie by Src. It will be interesting to elucidate how non-cell autonomous signaling is regulated by Src activation in a future study (Nishida, 2021).

Second, although Src was known to induce apoptosis as well as cell proliferation, how Src accomplishes this was unclear. This study elucidated that, diverging from Slpr, p38 accelerates cell proliferation and that JNK induces cell death. This is an obligatory coupling of proliferation and death, likely being accomplished through evolution as an imperative mechanism to prevent tumorigenesis by a single oncogene activation. This type of fail-safe mechanism to prevent facile transformation was previously suggested in a context of Myc oncogene. It is proposed that, although each oncogene should have its unique fail-safe mechanism, the concept of the intrinsic fail-safe mechanism to prevent oncogenesis by a single oncogene is general (Nishida, 2021).

Third, from a therapeutic perspective, the observation that methionine strongly regulates Src-mediated overgrowth is intriguing. Tumor growth in vitro is metabolically regulated by nutrition and dietary manipulation of serine, glycine, histidine, asparagine, cysteine, or methionine could clinically modulate cancer outcome. Notably, in the physiological in vivo condition, only subtraction of methionine from diet enhances organismal survival over Src-mediated oncogenic stress. Methionine has been studied in contexts of life span, metabolic health, and cancer together with other amino acids, but the molecular mechanisms behind methionine-mediated cellular and organismal physiology were often unclear. This study demonstrates that methionine regulates Tor activation, which controls cell proliferation induced by Src-p38 signaling (Nishida, 2021).

This study also found that the methionine concentration in the hemolymph is lower in flies that bear tumors in the wing disc. This is reminiscent of the clinical condition where tumor affects the amino acid profiles in the blood. Of note, local glutamine is known to be consumed in the tumor environment, but at least reduction of glutamine in the hemolymph of the flies bearing tumors was not observed. It is presumed that Src-induced increase of methionine uptake in the Src tumor is at least partly responsible for the Src tumor-induced hypomethioninemia, although other tissues may also contribute to it as the case with the fat body during wing disc repair (Nishida, 2021).

Regarding a cross-talk between Src signaling and nutrition-mediated Tor activation, this study found that there are multiple cross-talk points. Src regulates methionine uptake and methionine flux in a p38-independent manner, both of which can potentially feed into Tor activation. Then, a question is how Src-p38 regulates Tor signaling, since Src-p38 clearly activates Tor signaling. Although p38 is known to regulate Tor, its exact molecular mechanism remains unclear. Using the previously published RNAseq data on Src tumor in the wing disc, expression levels of potential Tor regulators were surveyed and genes were selected that are affected by Src expression, including amino acid transporters and GATOR complexes. GATOR complexes regulate Tor through Rag GTPases. This study examined whether their expression is regulated by Src in a p38-dependent manner using RT-qPCR. Among the amino acid transporters and GATOR complex components examined, only pathetic (path), an SLC36 amino acid transporter that can transport multiple amino acids, was significantly induced by Src in a p38-dependent manner. Since Path can mediate amino acids-mediated Tor activation, it is speculated that Src-p38 could regulate Tor potentially through Path-mediated uptake of non-methionine amino acids (Nishida, 2021).

These findings have significant implications in the field of cancer therapeutics. As described in Introduction, SFK inhibitors have been clinically unsuccessful in spite of SFKs' contribution to tumorigenesis and metastasis. It is expected that the new insights this study provides on the Src tumorigenesis may help pave the way to cancer treatment. Furthermore, the data imply that nutritional state and tumorigenesis are closely linked. It is speculated that, in case of tumors with a high SFK activity, manipulation of dietary methionine may have a clinical benefit (Nishida, 2021).

Search PubMed for articles about Drosophila p38a

Adachi-Yamada, T., et al. (1999). p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis. Mol. Cell. Biol. 19(3): 2322-9. PubMed ID: 10022918

Adams, R. H., et al. (2000). Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 6: 109-116. PubMed ID: 10949032

Allen, M., et al. (2000). Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells. J. Exp. Med. 191: 859-870. PubMed ID: 10704466

Brook. M., et al. (2006). Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways. Mol. Cell Biol. 26: 2408-2418. PubMed ID: 16508015

Craig, C. R., Fink, J. L., Yagi, Y., Ip, Y. T. and Cagan, R. L. (2004). A Drosophila p38 orthologue is required for environmental stress responses. EMBO Rep. 5(11): 1058-63. PubMed ID: 15514678

Demontis, F., Patel, V. K., Swindell, W. R. and Perrimon, N. (2014). Intertissue control of the nucleolus via a myokine-dependent longevity pathway. Cell Rep 7(5):1481-94. PubMed ID: 24882005

Han, S. J., et al. (1998). Molecular cloning and characterization of a Drosophila p38 mitogen-activated protein kinase. J. Biol. Chem. 273(1): 369-74. PubMed ID: 9417090

Han, Z. S., et al. (1998). A conserved p38 mitogen-activated protein kinase pathway regulates Drosophila immunity gene expression. Mol. Cell. Biol. 18(6): 3527-39. PubMed ID: 9584193

Inoue, H. et al. (2001). A Drosophila MAPKKK, D-MEKK1, mediates stress responses through activation of p38 MAPK. EMBO J 20: 5421-5430. PubMed ID: 11574474

Li, Q., Zhang, X., Hu, W., Liang, X., Zhang, F., Wang, L., Liu, Z. J. and Zhong, Y. (2016). Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila. Proc Natl Acad Sci U S A 113(11): 3072-3077. PubMed ID: 26929354

Lim, H. Y., Bao, H., Liu, Y. and Wang, W. (2019). Select septate junction proteins direct ROS-mediated paracrine regulation of Drosophila cardiac function. Cell Rep 28(6): 1455-1470. PubMed ID: 31390561

Mihaly, J., et al. (2001). The role of the Drosophila TAK homologue dTAK during development. Mech Dev 102: 67-79. PubMed ID: 11287182

Nishida, H., Okada, M., Yang, L., Takano, T., Tabata, S., Soga, T., Ho, D. M., Chung, J., Minami, Y. and Yoo, S. K. (2021). Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila. Elife 10. PubMed ID: 33902813

Paricio, N., Feiguin, F., Boutros, M., Eaton, S. and Mlodzik, M. (1999). The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling. EMBO J 18: 4669-4678. PubMed ID: 10469646

Sano, Y., et al. (2005). Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-terminal kinase. Mol. Biol. Cell 16(6): 2934-46. PubMed ID: 15788564

Seisenbacher, G., Hafen, E. and Stocker, H. (2011). MK2-dependent p38b signalling protects Drosophila hindgut enterocytes against JNK-induced apoptosis under chronic stress. PLoS Genet. 7(8): e1002168. PubMed ID: 21829386

Sun, H., Nishioka, T., Hiramatsu, S., Kondo, S., Amano, M., Kaibuchi, K., Ichinose, T. and Tanimoto, H. (2020). Dopamine receptor Dop1R2 stabilizes appetitive olfactory memory through the Raf/MAPK pathway in Drosophila. J Neurosci. PubMed ID: 32102921

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date revised: 25 October 2023

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