p38a MAP kinase: Biological Overview | References
Gene name - p38a MAP kinase
Synonyms - p38a
Cytological map position - 95E5-95E5
Function - signaling
Keywords - stress response, heat shock, oxidative stress, starvation
Symbol - p38a
FlyBase ID: FBgn0015765
Genetic map position - 3R:19,976,133..19,977,996 [-]
Classification - Serine/Threonine protein kinases, catalytic domain
Cellular location - cytoplasmic
|Recent literature||Whon, T. W., Shin, N. R., Jung, M. J., Hyun, D. W., Kim, H. S., Kim, P. S. and Bae, J. W. (2017). Conditionally pathogenic gut microbes promote larval growth by increasing redox-dependent fat storage in high-sugar diet-fed Drosophila. Antioxid Redox Signal [Epub ahead of print]. PubMed ID: 28462587
Changes in the composition of the gut microbiota contribute to the development of obesity and subsequent complications that are associated with metabolic syndrome. This study demonstrate the inter-relationship between Drosophila and their resident gut microbiota under chronic high-sugar diet (HSD) conditions. Chronic feeding of an HSD to Drosophila resulted in a predominance of resident uracil-secreting bacteria in the gut. Axenic insects mono-associated with uracil-secreting bacteria or supplemented with uracil under HSD conditions promoted larval development. Redox signaling induced by bacterial uracil promoted larval growth by regulating sugar and lipid metabolism via activation of p38a mitogen-activated protein kinase. This study has identified a new redox-dependent mechanism by which uracil-secreting bacteria protect the host from metabolic perturbation under chronic HSD conditions. The results illustrate how Drosophila and gut microbes form a symbiotic relationship under stress conditions.
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
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-MEKK1ur36 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).
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).
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; p38bd27 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 p38bKR or p38bAGF 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 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).
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).
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
Mihaly, J., et al. (2001). The role of the Drosophila TAK homologue dTAK during development. Mech Dev 102: 67-79. PubMed ID: 11287182
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
Takatsu, Y. et al. (2000). TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development. Mol Cell Biol 20: 3015-3026. PubMed ID: 10757786
Tamura, K., et al. (2000). Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 102: 221-231. PubMed ID: 10943842
ter Haar, E., Prabhakar, P., Liu, X. and Lepre, C. (2007). Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer. J. Biol. Chem. 282: 9733-9739. PubMed ID: 17255097
Tian, L., Chen, J., Chen, M., Gui, C., Zhong, C. Q., Hong, L., Xie, C., Wu, X., Yang, L., Ahmad, V. and Han, J. (2014). The p38 pathway regulates oxidative stress tolerance by phosphorylation of mitochondrial protein IscU. J Biol Chem [Epub ahead of print]. PubMed ID: 25204651
date revised: 25 November 2014
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