Gene name - p38b MAP kinase
Cytological map position - 34C4--D1
Function - protein kinase
Symbol - p38b
FlyBase ID: FBgn0024846
Genetic map position -
Classification - MAP kinase
Cellular location - probably both nuclear and cytoplasmic
|Recent literature||Huang, D., Li, X., Sun, L., Huang, P., Ying, H., Wang, H., Wu, J. and Song, H. (2016). Regulation of Hippo signalling by p38 signalling. J Mol Cell Biol [Epub ahead of print]. PubMed ID: 27402810
The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yorkie/YAP/TAZ by Warts/Lats. This study identifies the p38 MAPK pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts It was further demonstrated that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jelly belly phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, p38 signalling was shown to regulate Hippo signalling in mammalian cell lines. The Lic homolog MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. This work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation.
|Eom, H. J., Liu, Y., Kwak, G. S., Heo, M., Song, K. S., Chung, Y. D., Chon, T. S. and Choi, J. (2017). Inhalation toxicity of indoor air pollutants in Drosophila melanogaster using integrated transcriptomics and computational behavior analyses. Sci Rep 7: 46473. PubMed ID: 28621308
This study conducted an inhalation toxicity test on the alternative animal model, Drosophila melanogaster, to investigate potential hazards of indoor air pollution. The inhalation toxicity of toluene and formaldehyde was investigated using comprehensive transcriptomics and computational behavior analyses. The ingenuity pathway analysis (IPA) based on microarray data suggests the involvement of pathways related to immune response, stress response, and metabolism in formaldehyde and toluene exposure based on hub molecules. A toxicity test was conducted using mutants of the representative genes in these pathways to explore the toxicological consequences of alterations of these pathways. Furthermore, extensive computational behavior analysis showed that exposure to either toluene or formaldehyde reduced most of the behavioral parameters of both wild-type and mutants. Interestingly, behavioral alteration caused by toluene or formaldehyde exposure was most severe in the p38b mutant, suggesting that the defects in the p38 pathway underlie behavioral alteration. Overall, the results indicate that exposure to toluene and formaldehyde via inhalation causes severe toxicity in Drosophila, by inducing significant alterations in gene expression and behavior, suggesting that Drosophila can be used as a potential alternative model in inhalation toxicity screening.
Mitogen-activated protein kinase (MAPK) proteins are conserved eukaryotic factors that are integral to various signal transduction pathways. Three subgroups of the MAPK superfamily have been identified: the extracellular signal-regulated kinase (ERK); c-Jun N-terminal kinase (JNK, also referred to as stress-activated protein kinase [SAPK]), and p38 (also called Mpk2). MAPKs are activated through phosphorylation by specific MAPK kinases (MAPKKs or MEKs), which are themselves phosphorylated and activated by specific MAPKK kinases (MAPKKKs), thus comprising a series of separate MAPK cascades. Both ERK and JNK, termed Rolled and Basket, respectively, have been extensively characterized in Drosophila, but Drosophila p38 has been all but neglected. This neglect has now come to an end for several reasons: (1) two p38 homologs have now been cloned; (2) two MEKs (MAPK kinases) functioning immediately upstream of p38 have also been cloned; (3) a role for one of these MEKs, called Licorne, has been identified in oogenesis; (4) potential roles for these p38s and MEKs in the immune response have also been identified, and (5) in the study described in this overview, p38b is involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis (Adachi-Yamada, 1999).
Two Drosophila homologs of p38, Mpk2 (also known as p38a or simply p38) and p38b, have been identified on the basis of their homology to mammalian p38 and to one another (S. Han, 1998 and Z. Han, 1998). p38b, the subject of this overview, is maternally expressed and is present ubiquitously during embryonic development (Z. Han, 1998). A second approach, in addition to that of sequence homology, was also taken when cloning p38s in Drosophila. p38b was cloned on the basis of its ability to complement a defect in MAPK in yeast. The yeast hog1 pathway mediates cellular responses to an increase in external osmolarity (Herskowitz, 1995). Mammalian p38 has been shown to complement the high osmolarity-sensitive growth phenotype of the hog1 MAPK mutant. To isolate cDNAs for the Drosophila homolog(s) of p38, genetic screens were used to isolate suppressors of the hog1delta mutation. A total of 16 positive clones were obtained and assigned to either of two classes. The cDNAs from one class were identical to Drosophila Basket/JNK. cDNAs from the other class were identical to p38b. Comparison of the cDNA and genomic sequences revealed that the D-p38b gene is organized into two exons, although its coding region is continuous within a single exon (Adachi-Yamada, 1999).
The chromosomal region around the p38b locus has been well characterized genetically. However, a p38b transgene was unable to rescue any of the known mutations mapping to this region. Likewise, attempts to isolate a mutant of p38b were unsuccessful. These failures are possibly due to the functional redundancy of the two p38 homologs. Various alternative methods were therefore use to interfere with endogenous p38(s) in order to investigate its function. A dominant-negative allele of p38b, designated D-p38bDN, was generated by replacing the Thr-183 of the MAPKK target site with Ala, analogous to the change in ERK2 that produces a dominant-negative allele. This recombinant mutant protein lost its ability to suppress the yeast hog1delta mutant phenotype (Adachi-Yamada, 1999).
Two lines were prepared which express D-p38bDN at different levels: D-p38bDN-S (Strong), which expresses high levels, and D-p38bDN-W (Weak), which expresses low levels. When two copies of the D-p38bDN-S transgene are expressed in the wing, a certain fraction of adult flies that escape death exhibit ectopic vein fragments around the end of the longitudinal vein L2 and a reduction in the distance between L4 and L5. Both of these features have also been observed with some mutant alleles of decapentaplegic (dpp) and thick veins (tkv), a gene encoding a type I receptor for Dpp. This wing phenotype is rescued by coexpression of the wild type p38b+ transgene. When two copies of the D-p38bDN-S transgene are weakly expressed in the wing of a dpp mutant, the vein phenotype of dpp is strongly enhanced. These phenotypes suggest the involvement of Drosophila p38(s) in Dpp function in the early and late stages of wing pattern development. Dpp is known to play a dual role during wing development, acting as a morphogen and mitogen at early stages, while activating vein differentiation at later stages (Adachi-Yamada, 1999).
To examine whether p38(s) functions in the Dpp signaling pathway, the genetic interaction was examined between p38(s) and a constitutively active mutant of Tkv (TkvCA). Two classes of tkvCA insertions, tkvCA-S (Strong) and tkvCA-W (Weak), were used. When tkvCA-S is expressed, normal wing venation is severely distorted and extensive production of fragments of vein material is observed. The abdominal-cuticle pattern also appears irregular. This wing phenotype suggests that TkvCA may influence Dpp action during vein formation. Ectopic coexpression of dpp+ and tkv+ causes similar phenotypes, indicating that these TkvCA-induced aberrations are indeed the result of an increase in Dpp signaling. It was thus expected that reducing the levels of downstream components would suppress tkvCA. In fact, reducing by one-half the gene dosage of Mothers against dpp (Mad), a well-documented Dpp-signaling factor, significantly suppresses the tkvCA wing phenotype (Adachi-Yamada, 1999).
The effect of the imidazole compound SB203580, a p38 inhibitor, was tested on the tkvCA wing phenotype. SB203580 has been reported to inhibit both p38a and p38b (Z. Han, 1998), and penetration of various imidazole compounds through the insect epidermis is well known. Exposure of growing larvae to SB203580 indeed results in suppression of the phenotype. Tests were performed to see whether endogenous p38 genes are involved in the tkvCA wing phenotype by reducing endogenous gene dosage using chromosomal hemizygosity. Interestingly, reduction of p38b suppresses the tkvCA wing phenotype, while reduction of p38a is not effective. Suppression by reduction of the p38b gene dosage is abrogated by the introduction of a transgene for p38b+. Thus, the gene within the deficiency that suppresses tkvCA is indeed p38b. These results suggest that p38b plays a major role in this morphogenetic process, and attention was focussed on this gene in further analyses (Adachi-Yamada, 1999).
Antisense RNA can often be used to mimic the effects of mutation. When antisense p38d RNA is coexpressed with TkvCA-S, the tkvCA-S phenotype is markedly suppressed. Four of five independently established p38bantisense lines showed significant suppression. Suppression affects the various pleiotropic phenotypes associated with the tkvCA allele, including wing blade morphology, abdominal-cuticle morphology, and wing posture. Similar suppression of the tkvCA phenotype is also achieved by coexpression of p38bDN-W in a dose-dependent manner. This suppression is greater when the strong dominant negative p38b (p38bDN-S) is coexpressed instead of p38bDN-W. Furthermore, this suppression is abrogated by simultaneous coexpression of wild type p38d, demonstrating that p38bDN and wild type p38b competitively sequester endogenous factors essential to signaling. These results suggest either that p38b functions downstream of Tkv or that inhibition of p38b causes a reduction in endogenous dpp activity. Since the expression pattern of dpp in the developing wing of the D-p38bDN-S producer has been found to be indistinguishable from that of the wild type, and reduction in the gene dosage of dpp is not effective in suppressing the tkvCA phenotype, it is concluded that p38b does not affect Dpp production per se but rather acts as a downstream component of the Dpp-Tkv signaling pathway, operating late in wing development. The fact that the weak phenotype of tkvCA-W is significantly enhanced by wild type p38b is also consistent with this conclusion (Adachi-Yamada, 1999).
The effect of p38b on optomotor blind transcription was examined in order to study the involvement of p38b in the Dpp signaling pathway. The omb gene encodes a T-box family transcription factor, and its expression in the wing imaginal disc is dependent on early Dpp-Tkv signaling. In the wing discs of flies ectopically expressing tkvCA-5, the omb expression domain is greatly expanded and overgrowth of the disc is evident. Expression of p38bDN or p38bantisense markedly suppresses both omb expression and disc overgrowth. Induction of omb in the tkvCA-expressing clones in regions outside those where dpp is expressed is also inhibited by coexpression of p38bDN, consistent with the possibility that p38b functions downstream of Tkv. Furthermore, while p38bDN slightly affects omb expression in a tkv+ genetic background, the wing phenotype of a hypomorphic omb allele is clearly enhanced by expression of p38bDN or p38bantisense, as observed in the wing phenotype of severe omb alleles. These results suggest that p38b is also involved in early Dpp-Tkv signaling in wing development to activate omb transcription. Evidence is presented that p38b, or possibly both p38s, are phosphorylated in vivo downstream of ectopically expressed constitutively active Tkv (Adachi-Yamada, 1999).
To investigate whether p38b is activated by Tkv signaling, a preliminary biochemical characterization of p38b was carried out. Immediately after heat treatment of flies, the amount of p38b immunoprecipitated by anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected to be in the 'activation loop' region recognized by MAPKK, as is the case in mammalian p38. Thus, a test was performed to see whether an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of mammalian p38 could cross-react with p38b. This anti-p-p38 antibody detects a protein with a calculated size of 42 kDa whose amount increases immediately after heat shock. This protein is also more abundant in the flies overproducing p38b regardless of heat treatment. Therefore, it has been concluded that anti-phospho-p38 can cross-react with the phosphorylated from of p38b and can be used to assay recombinant p38b phosphorylation in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38 antibodies, and a drastic increase in the level of Drosophila p38-dependent phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for mammalian p38. The correlation between the phosphorylation state and kinase activity of p38b indicates that the anti-p-p38 antibody recognizes the active form of p38b. This allowed activation of p38b by TkvCA to be examined in vivo. The amount of active p38b was found to be slightly but significantly higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in yeast, which was presumed to have the same molecular mass as p38b, is also recognized by anti-p-p38 antibody. It is therefore possible that p38b, or both D-p38's, may be activated by Tkv signaling in vivo (Adachi-Yamada, 1999).
A p38 DNA sequence deposited by the Drosophila Genome Project was identified by searching GenBank with the p38a (Mpk2) sequence. This genomic DNA sequence contains an open reading frame (p38b), which also does not contain an intron and encodes a putative protein of 365 amino acids. The deduced amino acid sequence is 75% identical to that of p38a (Z. Han, 1998).
date revised: 30 July 99
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