BACKGROUND
mitogen-activated protein kinases  are serine-threonine protein kinases that regulate a variety of cellular processes, including growth, metabolism, apoptosis, and innate immune responses  <cit> .
mapks function in multi-tiered signaling cascades, in which an activated map4k phosphorylates and activates a map3k which, in turn, activates a downstream map2k, which activates a mapk that can regulate effector proteins or transcription factors to positively or negatively regulate suites of genes  <cit> .
mapk signaling modules provide multiple levels of regulation that confer signal amplification and specificity toward a desired outcome  <cit> .
a wide assortment of stimuli activate mapks, including inflammatory cytokines  <cit> , osmotic stress  <cit> , oxidative stress and redox signaling  <cit> , and growth factors  <cit> .
mapks have been extensively studied and a wealth of information is available from many model systems, including caenorhabditis elegans, drosophila melanogaster and a variety of mammals  <cit> .
from an evolutionary standpoint, mapks have diverged very little over time and several published phylogenies of mapks have revealed a high degree of conservation from invertebrates to vertebrates  <cit> .
further, these analyses have contributed to our understanding of the evolution and function of the mapks  <cit> .
for example, a mapk phylogeny was constructed from the encoded sequences in the genome of the human pathogenic blood fluke, schistosoma japonicum, together with known eukaryotic mapks from model organisms to elucidate putative functions of previously undescribed s. japonicum mapks  <cit> .
the construction of mapk phylogenies can, therefore, facilitate predictions of the roles of mapks in non-model organisms, including those of public health importance.
malaria is a parasitic disease of great public health concern, with over  <dig> million new cases per year, resulting in nearly one million deaths annually  <cit> .
in sub-saharan africa, the mosquito anopheles gambiae transmits the most deadly human malaria parasite plasmodium falciparum.
despite highly efficient transmission, the invertebrate and vertebrate hosts of malaria parasites can mount sophisticated immune responses to infection.
these responses are regulated in both hosts, in part, by mapks  <cit> .
two prominent parasite-derived signals - glycosylphosphatidylinositols  and hemozoin - activate mapk signaling in both the mammalian and mosquito hosts.
mammalian jnk, erk and p <dig> mapks transduce signals from p. falciparum glycophosphatidylinositols  for inflammatory cytokine synthesis in immune cells in vitro and during parasite infection in vivo  <cit> .
hemozoin signals principally through erk to increase interferon-gamma-dependent production of anti-parasite nitric oxide  in mammalian cells  <cit> .
in an analogous fashion, pfgpis function as an early signal of parasite infection in an.
gambiae  <cit>  and in anopheles stephensi  <cit> , a vector of malaria in asia and close relative of an.
gambiae.
in an.
stephensi, pfgpis robustly activate mek-erk phosphorylation in the mosquito midgut epithelium  <cit> , a site that is critical for parasite development in the insect host.
as in mammalian cells, hemozoin can activate mek-erk signaling in the an.
stephensi midgut  <cit> .
further, transforming growth factor -beta1-dependent mek-erk-dependent signaling can facilitate p. falciparum development at the midgut epithelium by inhibiting the expression of no synthase  <cit>  and synthesis of inflammatory levels of reactive nitrogen oxides that limit parasite development  <cit> .
in contrast to our understanding of erk signaling in an.
stephensi and an.
gambiae, our knowledge of the regulatory ligands and signaling pathways as well as the biological impacts of jnk and p <dig> mapk signaling in these species is more limited.
in particular, jnk activation appears to mediate as yet unidentified inhibitory responses to the murine parasite plasmodium berghei in an.
gambiae  <cit> , although the extracellular signals and upstream regulatory proteins for jnk activation are unknown.
in the case of p <dig> mapk, this signaling protein appears to regulate antimicrobial responses in aedes mosquitoes  <cit>  and p <dig> mapk is activated during insulin-dependent immune signaling in an.
stephensi  <cit> .
however, the pathway elements that regulate this signaling, the biological effects of signaling, and the identity of other activating ligands in anopheles spp., including those derived from malaria parasites  <cit> , are unknown.
in this study, we used bioinformatics, existing knowledge of mammalian signaling pathways, and signaling assays in an.
gambiae cells to provide a more comprehensive understanding of the likely roles of the mapk cascades in innate immunity of an.
gambiae, a critical malaria vector mosquito for which an annotated genome sequence is available  <cit> .
the mapk phylogeny presented here recapitulates the conservation of the mapks and, for the first time, identifies the orthologous associations and evolutionary origins of the complete suite of an.
gambiae mapks.
the functional data presented herein demonstrate differing roles for an.
gambiae p <dig> mapk, erk and jnk in response to stimuli that are biologically relevant for malaria parasite infection and, together with existing knowledge from mammalian models, suggest that anopheline mosquitoes utilize a unique mapk architecture for signaling.
methods
mapk identification
the initial set of  <dig> human mapks used for reference in this study was identified in the genecards database  <cit>  .
fifteen an.
gambiae mapk orthologs were identified in genecards and confirmed using basic local alignment search tool   <cit>  against an.
gambiae genome sequence data.
in addition, an.
gambiae sequence data were analyzed using highly conserved, orthologous mapk activation loop sequences.
the activation of map kinases occurs by a dual phosphorylation event by the upstream kinase on a solvent-exposed activation loop that typically contains a t-x-y motif  <cit> .
we used the encoded activation loop sequences from d. melanogaster slipper  and d. melanogaster wallenda , flanked by 5- <dig> additional conserved amino residues, in tblastn queries against an.
gambiae sequence data.
conserved catalytic domains for all predicted proteins were confirmed using prosite.
phylogenetic analysis
for phylogenetic analyses,  <dig> sequences, ranging from 115- <dig> amino acids, of mapk catalytic domains from an.
gambiae, c. elegans, ciona intestinalis, d. melanogaster, homo sapiens and saccharomyces cerevisiae were used to generate an alignment using the blosum protein weight matrix and clustalx  <cit> .
orthologs of c. elegans, d. melanogaster and c. intestinalis mapks were confirmed using blastp of genecards, ensemble metazoa, wormbase and flybase databases.
phylogenetic analyses were conducted using phylogeny inference package   <dig>   <cit> .
protdist was used to generate a distance matrix based on the jones-taylor-thornton  model of amino acid substitution.
a distance-based phylogenetic tree was then inferred using the neighbor-joining  algorithm implemented in neighbor.
seqboot was used to prepare  <dig> pseudoreplicate datasets for bootstrap analysis; these replicate datasets were then analyzed using protdist with the jtt model and neighbor to create a bootstrap nj tree.
the bootstrap majority-rule consensus tree was generated using consense, visualized in figtree v  <dig> .
<dig> http://tree.bio.ed.ac.uk/software/figtree/ and edited using inkscape  <dig>  http://inkscape.org/.
the unrooted consensus tree was drawn to scale, with branch lengths representing the mean number of substitutions per site.
mosquito cell culture and experimental treatments
the immortalized, embryo-derived an.
gambiae 4a3b cell line   <cit>  was maintained in schneider's medium  with 10% heat-inactivated fetal bovine serum  at 28°c.
for each condition,  <dig> ×  <dig> 4a3b cells in  <dig> ml medium were plated in one well of a 12-well tissue culture plate and allowed to recover overnight.
cells were then treated with the following stimuli at concentrations previously validated for signaling:  <dig> μm hydrogen peroxide   <cit> ,  <dig> pg/ml human tgf-beta <dig>   <cit> ,  <dig>  μm human insulin   <cit>  or  <dig> μg/ml lipopolysaccharide   <cit>  and collected at  <dig> min,  <dig> min,  <dig> min,  <dig> h,  <dig> h,  <dig> h and  <dig> h after treatment for subsequent western blot analysis.
to quantify and analyze erk, jnk, and p <dig> mapk transcript expression, 4a3b cells prepared as described above were treated with  <dig>  μm human insulin and collected at  <dig> h,  <dig> h,  <dig> h and  <dig> h for qpcr .
controls for each time point consisted of an identical aliquot of cells treated with diluent.
at least three biological replicates were performed for each treatment.
protein extraction and western blotting
protein extracts of 4a3b cells were prepared by collecting cells in lysis buffer as previously described  <cit> .
briefly, cell medium was removed following treatment and cells were washed with ice cold phosphate buffered saline  and lysed in the plate in  <dig> μl cell lysis buffer .
the plate was agitated for  <dig> min at 4°c and samples were incubated on ice for  <dig> min.
cell lysates were centrifuged at  <dig>  × g for  <dig> min at 4°c to remove cellular debris and  <dig> μl of supernatant from each sample was mixed with  <dig> μl of  <dig> × sample buffer  and heated at 95°c for  <dig> min.
protein samples were electrophoretically separated on 10% polyacrylamide gels via sodium dodecyl sulfate polyacrylamide gel electrophoresis  and transferred to nitrocellulose membranes .
protein loading was visually assessed by coomassie blue staining.
membranes were blocked in 5% nonfat dry milk  in  <dig> × tris-buffered saline containing  <dig> % tween- <dig>  for  <dig> h at room temperature.
for phosphorylated erk detection, membranes were incubated at 4°c overnight with 1: <dig>  mouse anti-phospho-erk monoclonal antibody  in 5% nfdm in tbs-t. for detection of phospho-p <dig>  phospho-jnk and the loading control protein glyceraldehyde 3-phosphate dehydrogenase , membranes were incubated at 4°c overnight with 1:  <dig>  rabbit anti-phospho-p <dig> mapk antibody , 1: <dig>  rabbit anti-phospho-jnk1/ <dig> antibody , or with 1: <dig>  rabbit anti-gapdh antibody  in 5% nfdm in tbs-t. membranes were washed  <dig> times for  <dig> min in  <dig> × tbs-t and incubated with a 1: <dig>  dilution of horse radish peroxidase -conjugated rabbit anti-mouse igg  or with a 1: <dig>  dilution of hrp-conjugated goat anti-rabbit  <dig> fragment  at 4°c overnight.
following incubation with the secondary antibody, membranes were washed  <dig> times for  <dig> min in  <dig> × tbs-t. to reveal antibody-bound proteins, membranes were incubated with supersignal west pico chemiluminescent reagent  for 2- <dig> min.
each membrane was exposed to blue autoradiography film .
phospho-mapk levels and total gapdh levels were quantified on scanned film using a gs- <dig> calibrated densitometer .
mapk quantitative pcr
primers for qpcr of an.
gambiae erk, p <dig>  jnka, and jnkb were designed using primer3plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi.
the primers included: erk, 5'atcccgagcacgatcaca3'  and 5'cgattttgtgtagcccttgga3' ; p <dig> mapk, 5'cggaccacattcaccagcta3'  and 5'cgctaaagttgcgcttctcc3' ; jnka, 5'gcacgcagcgatacattagc3'  and 5'gtccaccgaaatcctttcca3' ; and jnkb, 5'gaccgactcgaacgagcac3'  and 5'cgaaccacacgttgatgtagc3' .
the housekeeping gene encoding ribosomal protein s <dig> was analyzed as a control with the primers 5'gaaggccttccagaaggtacaga3'  and 5'catcggtttgggcagaatg3' .
rna was isolated from 4a3b cells using trizol regent  and contaminating dna was removed from the rna samples using turbo dna-free™ .
all rna samples were diluted to  <dig> ng/μl and  <dig> μl  rna from each sample was reverse-transcribed using superscript® iii .
sample cdnas were used to perform qpcr using maxima sybr green/rox qpcr master mix .
the qpcr cycling conditions were as follows:  <dig> min at 50°c,  <dig> min at 95°c, and  <dig> cycles of  <dig> s at 95°c and  <dig> min at 60°c.
three biological replicates with independent batches of 4a3b cells were analyzed for the diluent controls and treatments.
no template controls were included for each reaction plate and all reactions were performed in triplicate to confirm amplification consistency .
the triplicate data were used to determine an average ct for each reaction and the data analyzed using the 2-ΔΔct method as described  <cit> .
briefly, the average ct value for each target was first normalized to the average ct for ribosomal protein s <dig> gene to obtain Δct, and then the Δct value for each insulin-treated sample was normalized to timepoint-matched control to obtain ΔΔct.
fold-change of expression relative to the diluent control was determined using 2-ΔΔct.
statistical analyses
for western blotting data, the kolmogorov-smirnov test revealed that non-normal distributions were present in our data sets .
overall significance values of all data sets, therefore, were determined by the kruskal-wallis test and dunn's post test for pairwise comparisons .
for the q-pcr data, one way anova and student-neuman-keuls  were used for analyses of significance .
RESULTS
mapk discovery
we used a combination of bioinformatic techniques to identify a total of  <dig> mapks from the an.
gambiae genome .
among these, agap <dig> and agap <dig> were newly identified mapk orthologs.
based on homology, agap <dig> is most appropriately defined as an.
gambiae map3k10/ <dig> or mixed lineage kinase  2/ <dig> .
similarly, agap <dig> is most appropriately defined as an.
gambiae map3k 12/ <dig> .
agap <dig> and agap <dig> are annotated as p <dig> mapk and jnk orthologs, respectively, in vectorbase  <cit> .
however, inspection of agap <dig> by alignment with p <dig> mapk orthologs and reverse psi -blast for conserved domains revealed that the annotated sequence was missing key n-terminal residues in the kinase catalytic domain.
specifically, rps-blast of the annotated sequence predicted only  <dig> of  <dig> active site and  <dig> of  <dig> activation loop residues .
because the annotation appeared to be incomplete, we used the first  <dig> encoded amino acids from the n-terminus of the ae.
aegypti p <dig> mapk ortholog  to query the national center for biotechnology information  an.
gambiae trace archives database  and identified the probable n-terminus of agap <dig> .
similarly, the annotation of agap <dig> in vectorbase  <cit>  does not encode a start methionine.
approximately  <dig>  base pairs of sequence upstream of the 5'-most nucleotides of agap <dig> were translated in the three forward reading frames and analyzed by blastx using the first  <dig> amino acids of the corresponding orthologs from ae.
aegypti  and from culex quinquefasciatus .
a putative start methionine with kozak consensus and additional n-terminal amino acids extended the encoded sequence of agap <dig> .
the map4ks, the map3ks, the map2ks are present in 1: <dig> orthology with gene products that have been identified in d. melanogaster .
among the mapks, only d. melanogaster rolled and an.
gambiae erk are in 1: <dig> orthology.
in contrast, the d. melanogaster genome encodes two p <dig> mapk orthologs , a single jnk ortholog , an erk <dig> ortholog , and an erk <dig> ortholog, although the latter two are not associated with prototypical map kinase cascades  <cit> .
the an.
gambiae genome, on the other hand, encodes a single p <dig> mapk, two jnk orthologs , and no erk <dig> and erk <dig> orthologs.
based on sequence homology, an.
gambiae jnkb is most closely related to d. melanogaster basket .
the lack of strict 1: <dig> orthology among the mapks is perhaps not surprising because diversification among these proteins to accommodate unique aspects of host biology would have a greater likelihood of being tolerated relative to upstream changes in the signaling cascades that could result in significantly amplified non-adaptive phenotypes.
to determine whether the complement of mapks in the an.
gambiae genome was similar to that in other mosquito genomes, we examined the available erk, jnk, and p <dig> mapk orthologs for ae.
aegypti and cx.
quinquefasciatus .
the genome sequences of ae.
aegypti and cx.
quinquefasciatus are currently assembled into supercontigs in vectorbase  <cit>  .
in this context, if identical nucleotide sequences  are excluded, erk, jnka, and jnkb orthologs are in 1:1: <dig> orthology among the three mosquito species.
in contrast, cx.
quinquefasciatus appears to possess two relatively short mapks that are tentatively identified as p <dig> mapks.
the encoded sequences share significant identity to ae.
aegypti p <dig> mapk and the putative full-length an.
gambiae p <dig> mapk, but are non-overlapping sequences, so it is impossible to discern whether these are indeed truly distinct gene products encoded in the cx.
quinquefasciatus genome.
because these relationships cannot be confirmed with the existing genome assemblies, the ae.
aegypti and cx.
quinquefasciatus mapk sequences were excluded from the phylogenetic analysis.
phylogeny
phylogenetic analysis of the mapk catalytic domains  showed strong support for the map4k and map2k groups .
the map3k raf superfamily formed a group , but the map3k mekk family did not form an exclusive group.
similarly, the two map3k superfamilies did not group together as an exclusive group and the sequences representing mapk were not supported as a group in this analysis.
the lack of a close relationship between the raf and mekk map3k groups was not unexpected based on previous phylogenetic analyses  <cit> .
the mekk family proteins  and the raf superfamily proteins  are both associated with p <dig> and jnk signaling pathways; however, raf proteins also figure prominently in mek/erk signaling   <cit> .
although not reflected in this analysis, the previously noted division of the erk subfamily  may be due to the classification of erk <dig>   <dig> and 7/ <dig> as mapk-like cascade independent kinases  <cit> .
frequently, sequences from the two deuterostomes  and the insects  grouped together.
exceptions to these groupings occurred when there were no clear homologs , which likely reflects the sequence diversification that has resulted in the many mapks that are found in humans relative to other species.
functional assays
the identification of the complement of map4ks, map3ks, map2ks and mapks provides a framework for the prediction of patterns of activation of cell signaling cascades in an.
gambiae cells.
to attempt to determine whether these pathways are phenotypically represented in this mosquito species, we selected three stimuli  that we had previously implicated in erk- and p <dig> mapk-dependent an.
gambiae immune signaling  <cit> , as well as lipopolysaccharide , an activator of immune signaling pathways in mosquito cells  <cit> , to stimulate an.
gambiae 4a3b cells over an extended timecourse from  <dig> min to  <dig> h. a representative western blot is shown in additional file  <dig>
stimulation with  <dig> μm hydrogen peroxide induced significant activation of p <dig> mapk at  <dig> min relative to the control group .
signaling declined from  <dig> min to  <dig> h post-stimulation, with a return to control levels by  <dig> h .
despite trends toward induction within  <dig> h of stimulation, erk and jnk were not significantly activated by  <dig> μm hydrogen peroxide in 4a3b cells .
in previous studies, stimulation of 4a3b cells with  <dig> μm hydrogen peroxide induced activation of p <dig> mapk as well as erk by  <dig> min post-treatment  <cit> , a faster and more extensive mapk induction than observed here for  <dig> μm hydrogen peroxide .
in accord with previous observations at  <dig> min post-treatment  <cit> , stimulation with  <dig>  μm human insulin induced significant activation of erk and p <dig> mapk at  <dig> min relative to the control and  <dig> min time point .
in contrast to the more temporally limited activation by hydrogen peroxide, insulin-activated erk and p <dig> mapk were detectable to  <dig> h and  <dig> h post-stimulation, respectively, suggesting a greater persistence of these signaling responses.
phosphorylation of jnk was slightly, although not significantly, elevated relative to control levels within  <dig> h after stimulation .
stimulation with  <dig> pg/ml human tgf-beta <dig> induced significant activation of erk only at  <dig> min post-treatment relative to the  <dig> min time point , with detectable levels of activated erk through  <dig> h post-stimulation.
this induction of erk activation at  <dig> min, together with a lack of p <dig> mapk activation, was noted previously  <cit> .
in contrast to slight trends toward jnk activation by hydrogen peroxide and insulin , phosphorylated jnk levels never exceeded baseline control levels at all times after stimulation with human tgf-beta <dig> .
stimulation with  <dig> μg/ml lps presented a unique pattern of mapk activation in an.
gambiae 4a3b cells.
in contrast to erk and p <dig> mapk activation by hydrogen peroxide, insulin and tgf-beta <dig> , lps stimulation did not induce mean phosphorylated erk levels above baseline  and only non-significant trends toward elevated phosphorylated p <dig> mapk were noted through  <dig> min post-stimulation .
levels of phosphorylated jnk, however, were significantly increased by lps stimulation  at  <dig> min and  <dig> min post-treatment relative to control, with slightly elevated but not significant levels of phosphorylated jnk through  <dig> h post-treatment that declined to baseline control levels by  <dig> h post-treatment.
although the majority of signaling protein genes do not exhibit transcriptional responsiveness to environmental stimuli , we questioned whether we might detect a signaling response at the mapk transcript level in an.
gambiae cells.
to address this question, we used qpcr to analyze expression levels of p <dig> mapk, jnka, jnkb, and erk expression in response to human insulin, the only stimulus for which we detected changes in phosphorylation for more than one mapk .
in replicated assays, none of the four genes exhibited a significant change in expression from  <dig> h to  <dig> h after treatment with  <dig>  μm human insulin .
together with our phosphorylation data, these data suggest that mapk responsiveness in an.
gambiae cells occurs principally at the protein level.
discussion
the protein targets of the mammalian mapks   <cit>  predict a strong association of the mapks with immune regulation.
this association is also ancient, with strong representation in invertebrates.
in c. elegans, p <dig> mapk- and perk-dependent signaling pathways, as well as insulin and tgf-beta signaling pathways, are required for host defense, with the general presumptions that insulin signaling functions in pathogen surveillance, p <dig> mapk-dependent signaling functions in general defense, and p <dig> mapk-independent signaling pathways provide pathogen-specific responses .
this nematode, however, lacks orthologs of rel and nuclear factor -kappab, the primary immune signaling transcription factors of d. melanogaster.
in the fruit fly, toll- or imd-dependent signaling regulates nf-kappab and jnk pathways, while p <dig> mapk-dependent signaling mediates host defense against pathogen invasion  <cit> .
the jnk and p <dig> mapks also mediate control of antimicrobial peptide expression in d. melanogaster  <cit> , but cooperativity with nf-κb appears to be restricted to jnk.
in particular, imd activation results in the activation of tak <dig> upstream of jnk and i-kappa kinase , the upstream activator of rel that ultimately feeds back to reduce jnk activation  <cit> .
the complement of map kinases encoded in the an.
gambiae genome would provide this organism with the significant redundancy of p <dig> mapk and jnk signaling observed in model organisms, although some intriguing differences between the mosquito and other species are predicted as well.
in particular, an.
gambiae map4k <dig> may function similarly to its mammalian counterpart downstream of toll and rac <dig>  to activate the mosquito mlk2/ <dig> ortholog  and p <dig> mapk and jnk signaling .
the second an.
gambiae map4k - map4k <dig> - would be predicted to activate map3k <dig> and map3k <dig>  however, an.
gambiae lacks an ortholog for map3k <dig>  an upstream activator for mek/erk signaling, so map4k <dig> activation of map3k <dig>  would be predicted to activate nf-kappab signaling through ikk , and p <dig> mapk and jnk signaling through map2k <dig> and map2k <dig> activation, respectively.
among the other an.
gambiae map3k orthologs, a number are predicted to lack well-defined upstream activators or to function independently of map4k activation.
in particular, the activators of an.
gambiae ortholog of map3k12/ <dig>  are not well defined based on data from mammalian models ; nevertheless, the mosquito protein would be predicted to contribute to the activation of jnk signaling .
among the map3k orthologs that can be activated independently of map4ks, an.
gambiae map3k <dig> would be predicted to lie downstream of tgf-beta activation of gadd <dig> , while map3k <dig> could be activated by tgf-beta-mediated activation of daxx  or tnf/tnfr activation of traf <dig> .
eiger and wengen have been identified as d. melanogaster orthologs of tnf/tnfr and, in the context of innate immunity, eiger functions independently of the toll and imd signaling pathways to regulate pathogen-induced cell death  <cit> , proliferation of extracellular pathogens  <cit>  and prophenoloxidase-mediated defense responses  <cit> .
although a clear ortholog of traf <dig> is not apparent in the an.
gambiae genome, orthologs of eiger  and wengen  would suggest that signaling by this ligand and receptor could lead to map3k5/map2k <dig> activation of p <dig> mapk.
an examination of possible pathways leading to mek/erk signaling based on the encoded map kinases reveals that, in addition to a lack of map3k <dig>  an.
gambiae also lacks an ortholog for map3k <dig>  so the regulation of mek/erk signaling is likely dependent on b-raf and mos activation, which are the downstream targets for rap <dig>  and for ras , a major transducer for multiple signaling inputs from growth factors and cytokines .
the data from our functional studies  not only confirm a subset of signaling predictions from known pathways , but also suggest that other pathways may be involved in an.
gambiae mapk activation.
in particular, human insulin-induced activation of an.
gambiae erk was predicted  and this was consistent with previous observations  <cit> .
in contrast, activation of an.
gambiae p <dig> mapk by insulin, which may occur through hydrogen peroxide at a concentration that is insufficient for erk activation , is not predicted to flow through map4k-map3k-map2k activation .
interestingly, several studies have reported cross talk between the insulin receptor kinase and the janus-activated kinase  signaling pathways  <cit> .
in particular, jak <dig> - the major mediator of inflammatory cytokine signaling in mammalian cells - can be activated by hydrogen peroxide  <cit>  and contributes to insulin-dependent activation of p <dig> mapk, jnk, and erk in a manner that is independent of insulin receptor substrate activation and activation of the phosphatidylinositol 3-kinase/akt-dependent arm of insulin signaling  <cit> .
jaramillo-gutierrez et al.
<cit>  proposed, based on the regulation of antioxidant gene expression by jnk, that this mapk may in fact be induced by oxidative stress to control redox biology in an.
gambiae.
these observations - together with our data and known models of mapk signaling  - predict the existence of a pathway involving insulin-induced hydrogen peroxide regulation of a jak ortholog  and downstream activation of p <dig> mapk and jnk that can be tested experimentally in an.
gambiae.
in contrast to the prediction of erk-specific signaling by insulin, existing mammalian signaling data  predict that human tgf-beta <dig> should activate erk as well as p <dig> mapk in an.
gambiae cells.
we have extensively characterized tgf-beta1-dependent erk signaling in the regulation of p. falciparum development in an.
stephensi  <cit>  and we have also demonstrated that p <dig> mapk is activated by tgf-beta <dig> in an.
stephensi cells  <cit> .
given that p <dig> mapk can be activated in an.
gambiae cells , our understanding of mammalian mapk signaling  would suggest that perhaps a deficiency in the tgfbr-gadd45-map3k <dig> cascade is responsible for the failure of tgf-beta <dig> to activate p <dig> mapk in an.
gambiae 4a3b cells.
a comparative analysis of an.
stephensi and an.
gambiae 4a3b cells, therefore, could be used to identify critical differences in these signaling pathways.
lps has been used as an activator of a variety of immune signaling pathways in mosquito cells  <cit> .
a putative an.
gambiae rac1-map4k1-map3k11-map2k7-jnk signaling cascade  could provide one mechanism for lps activation of jnk in an.
gambiae 4a3b cells .
however, we could also predict, given that this mosquito map3k is orthologous with map3k <dig> as well as map3k <dig> that lps should also activate p <dig> mapk signaling through map2k <dig>  because lps does not significantly activate p <dig> mapk in an.
gambiae 4a3b cells , we could speculate that this an.
gambiae map3k is functionally more analogous to map3k <dig>  an examination of map2k <dig> and map2k <dig> activation downstream of an.
gambiae map3k10/ <dig> using inhibitors or knock down strategies could clarify the orthology and functionality of this mosquito map3k.
although lps was the only pathogen-associated molecular pattern  used in these signaling studies, the restriction of mapk activation by lps to an.
gambiae jnk is insightful.
akman-anderson et al.
<cit>  showed that p. falciparum hemozoin induced activation of tak <dig> and erk as well as the signaling kinases akt/protein kinase b and atypical protein kinase c zeta/lambda in an.
gambiae 4a3b cells.
activation of tak <dig> by this important parasite signaling factor, together with lps-specific activation of jnk in our studies, suggests that jnk may function specifically in pathogen detection in an.
gambiae cells.
indeed, following infection with p. berghei or p. falciparum, the mosquito midgut epithelium undergoes profound cytoskeletal changes similar to those regulated by the toll/map2k4/jnk signaling module in d. melanogaster  <cit> .
additional support of the importance, and perhaps specificity, of jnk signaling for control of natural p. falciparum infection in an.
gambiae is provided by two population genetics studies.
we identified a single nucleotide polymorphism  in the an.
gambiae map2k <dig>  gene that was in linkage disequilibrium with a snp in the gene encoding toll5b that was significantly associated with p. falciparum infection  <cit> .
this synonymous snp, mkk <dig>  introduces a reduction of codon frequency greater than 3-fold, which could result in changes in downstream protein expression or function and, hence, jnk signaling  <cit> .
in addition to our work, riehle et al.
<cit>  mapped a locus that is strongly associated with an.
gambiae resistance to p. falciparum infection in east africa that encompasses the chromosomal locations of map4k <dig> and map3k <dig>  both of which are predicted to regulate jnk signaling .
CONCLUSIONS
in sum, we have identified the complement of an.
gambiae map kinases and used a combination of predicted signaling relationships and bioassays to suggest novel interactions and functionality that can be tested experimentally in this biomedically important mosquito species.
clearly, the mapk signaling cascades are the major regulators of innate immunity in c. elegans, in d. melanogaster, and in mammals, but we are only beginning to appreciate the importance of these complex cascades in innate immunity of the mosquito genera that serve as vectors of globally devastating pathogens of humans.
the establishment of this "road map" based on the most advanced mosquito genome annotation can, therefore, accelerate our understanding of host-pathogen interactions and broader physiology in an.
gambiae and in the closely related an.
stephensi and provide a reasonable architecture for similar efforts in ae.
aegypti and cx.
quinquefasciatus.
further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the map kinase cascades and other networked pathways , will ultimately be necessary to elucidate the "master signaling regulators" in these biomedically important insects.
list of abbreviations
ask: apoptosis signal-regulating kinase; blastx: basic local alignment search tool for translated query against a protein database ; daxx: death-associated protein 6; dlk: dual leucine zipper kinase; erk: extracellular signal-regulated kinase; gadd45: growth arrest and dna-damage inducible protein; gapdh: glyceraldehyde 3-phosphate dehydrogenase; hrp: horse radish peroxidase; ikk: i kappa b kinase; jak-stat: janus kinase/signal transducers and activators of transcription; jnk: jun kinase; lps: lipopolysaccharide; map4k: mitogen-activated protein kinase kinase kinase kinase; map3k: mitogen-activated protein kinase kinase kinase; map2k: mitogen-activated protein kinase kinase; mapk: mitogen-activated protein kinase; mek: equivalent to map2k; mekk: equivalent to map3k; mlk: mixed lineage kinase; nfdm: nonfat dry milk; nf-kappab: nuclear factor-kappab; no: nitric oxide; pfgpis: plasmodium falciparum glycosylphosphatidylinositols; prke1: equivalent to map3k <dig>  mlk1; rac1: ras-related c <dig> botulinum toxin substrate 1; rap1: ras-related protein 1; tak1: tgf-beta-activated kinase 1; tblastn: basic local alignment search tool for protein query against a translated database ; tbs-t:  <dig> × tris-buffered saline with  <dig> % tween 20; tgf-beta1: transforming growth factor-beta1; tnfr: tumor necrosis factor receptor; traf2:/6: tnfr-associated factor 2/ <dig>
authors' contributions
aah and msp confirmed the identities of the annotated and unannotated an.
gambiae mapks, co-wrote the initial draft, and assisted in the preparation of figures/files.
aah also edited the manuscript and revisions.
bw performed the mapk signaling and expression assays with an.
gambiae cells, analyzed the data, and edited the manuscript and revisions.
lc and san performed the phylogenetic analyses, prepared and edited the phylogram, and edited the manuscript.
aa, mn and jrf created the mapk signaling diagram based on the kegg pathway database and current literature and edited the manuscript.
sl conceived of this work, directed the studies, and co-wrote the initial and final drafts for publication.
all authors read and approved the final manuscript.
supplementary material
additional file 1
mapks used in this study.
h. sapiens, d. melanogaster, c. elegans and c. intestinalis are listed by common names and by accession number.
an.
gambiae mapks are listed by vectorbase agap numbers as well as by ncbi accession numbers and common names.
dashes indicate the absence of identified orthologs.
n/a for an.
gambiae map3k <dig> indicates no available ncbi accession number.
click here for file
additional file 2
alignment of predicted mosquito p <dig> mapks.
alignment of an.
gambiae , ae.
aegypti  and cx.
quinquefasciatus  p <dig> mapks.
the ae.
aegypti and cx.
quinquefasciatus sequences were used to query the an.
gambiae trace archives database to identify the probable n-terminal amino acids of an.
gambiae p <dig> mapk .
click here for file
additional file 3
alignment of an.
gambiae jnka and ae.
aegypti and cx.
quinquefasciatus jnk orthologs.
alignment of the first  <dig> amino acids of ae.
aegypti  and cx.
quinquefasciatus  jnk orthologs with upstream sequence from agap <dig> was used to predict the start methionine and additional n-terminal amino acids  of an.
gambiae jnka.
click here for file
additional file 4
predicted amino acid sequences of erk, jnk, p <dig> mapk orthologs from ae.
aegypti and cx.
quinquefasciatus.
ae.
aegypti and cx.
quinquefasciatus mapk orthologs as predicted from the supercontig assemblies for genome sequences for these species.
these sequences were excluded from the phylogeny and, therefore, the relationships of these orthologs with an.
gambiae mapks cannot definitively be confirmed.
click here for file
additional file 5
lps-induced mapk phosphorylation in an.
gambiae 4a3b cells.
cells were treated with  <dig> μg/ml lps or an equivalent volume of pbs.
cells were collected at  <dig> min,  <dig> min,  <dig> h,  <dig> h,  <dig> h and  <dig> h after treatment.
mapk phosphorylation was examined by western blotting as described in the methods.
gapdh levels provided an assessment of protein loading and were used to normalize corresponding phospho-mapk levels.
this figure is a representative of immunoblots from 6- <dig> independent experiments.
click here for file
additional file 6
mapk transcript expression in insulin-stimulated an.
gambiae 4a3b cells.
cells were treated with  <dig>  μm human insulin or an equivalent volume of diluent and collected at  <dig> h,  <dig> h,  <dig> h and  <dig> h after treatment .
expression levels of erk , p <dig> mapk , jnka , and jnkb  were analyzed by qpcr as described in the methods.
expression of ribosomal protein s <dig>  a housekeeping gene control, was used for normalization of treatment and control expression.
insulin-treated mapk expression levels are shown as fold changes relative to the timepoint-matched control group levels.
click here for file
acknowledgements and funding
we acknowledge support provided by hannah smithers, kong wai cheung and andrew ross for technical support for cell culture experiments and dr. ian korf for computational support.
this work was funded by the national institutes of health national institute of allergy and infectious diseases r <dig> ai <dig> and r <dig> ai <dig> to sl.

 mitogen-activated protein kinases are serine-threonine protein kinases that regulate a variety of cellular processes, including growth, metabolism, apoptosis, and innate immune responses. mapks function in multi-tiered signaling cascades, in which an activated map4k phosphorylates and activates a map3k which, in turn, activates a downstream map2k, which activates a mapk that can regulate effector proteins or transcription factors to positively or negatively regulate suites of genes. mapk signaling modules provide multiple levels of regulation that confer signal amplification and specificity toward a desired outcome. a wide assortment of stimuli activate mapks, including inflammatory cytokines, osmotic stress, oxidative stress and redox signaling, and growth factors. mapks have been extensively studied and a wealth of information is available from many model systems, including caenorhabditis elegans, drosophila melanogaster and a variety of mammals. from an evolutionary standpoint, mapks have diverged very little over time and several published phylogenies of mapks have revealed a high degree of conservation from invertebrates to vertebrates. further, these analyses have contributed to our understanding of the evolution and function of the mapks. for example, a mapk phylogeny was constructed from the encoded sequences in the genome of the human pathogenic blood fluke, schistosoma japonicum, together with known eukaryotic mapks from model organisms to elucidate putative functions of previously undescribed s. japonicum mapks. the construction of mapk phylogenies can, therefore, facilitate predictions of the roles of mapks in non-model organisms, including those of public health importance. malaria is a parasitic disease of great public health concern, with over million new cases per year, resulting in nearly one million deaths annually. in sub-saharan africa, the mosquito anopheles gambiae transmits the most deadly human malaria parasite plasmodium falciparum. despite highly efficient transmission, the invertebrate and vertebrate hosts of malaria parasites can mount sophisticated immune responses to infection. these responses are regulated in both hosts, in part, by mapks. two prominent parasite-derived signals - glycosylphosphatidylinositols and hemozoin - activate mapk signaling in both the mammalian and mosquito hosts. mammalian jnk, erk and p mapks transduce signals from p. falciparum glycophosphatidylinositols for inflammatory cytokine synthesis in immune cells in vitro and during parasite infection in vivo. hemozoin signals principally through erk to increase interferon-gamma-dependent production of anti-parasite nitric oxide in mammalian cells. in an analogous fashion, pfgpis function as an early signal of parasite infection in an. gambiae and in anopheles stephensi, a vector of malaria in asia and close relative of an. gambiae. in an. stephensi, pfgpis robustly activate mek-erk phosphorylation in the mosquito midgut epithelium, a site that is critical for parasite development in the insect host. as in mammalian cells, hemozoin can activate mek-erk signaling in the an. stephensi midgut. further, transforming growth factor -beta1-dependent mek-erk-dependent signaling can facilitate p. falciparum development at the midgut epithelium by inhibiting the expression of no synthase and synthesis of inflammatory levels of reactive nitrogen oxides that limit parasite development. in contrast to our understanding of erk signaling in an. stephensi and an. gambiae, our knowledge of the regulatory ligands and signaling pathways as well as the biological impacts of jnk and p mapk signaling in these species is more limited. in particular, jnk activation appears to mediate as yet unidentified inhibitory responses to the murine parasite plasmodium berghei in an. gambiae, although the extracellular signals and upstream regulatory proteins for jnk activation are unknown. in the case of p mapk, this signaling protein appears to regulate antimicrobial responses in aedes mosquitoes and p mapk is activated during insulin-dependent immune signaling in an. stephensi. however, the pathway elements that regulate this signaling, the biological effects of signaling, and the identity of other activating ligands in anopheles spp., including those derived from malaria parasites, are unknown. in this study, we used bioinformatics, existing knowledge of mammalian signaling pathways, and signaling assays in an. gambiae cells to provide a more comprehensive understanding of the likely roles of the mapk cascades in innate immunity of an. gambiae, a critical malaria vector mosquito for which an annotated genome sequence is available. the mapk phylogeny presented here recapitulates the conservation of the mapks and, for the first time, identifies the orthologous associations and evolutionary origins of the complete suite of an. gambiae mapks. the functional data presented herein demonstrate differing roles for an. gambiae p mapk, erk and jnk in response to stimuli that are biologically relevant for malaria parasite infection and, together with existing knowledge from mammalian models, suggest that anopheline mosquitoes utilize a unique mapk architecture for signaling. methods mapk identification the initial set of human mapks used for reference in this study was identified in the genecards database. fifteen an. gambiae mapk orthologs were identified in genecards and confirmed using basic local alignment search tool against an. gambiae genome sequence data. in addition, an. gambiae sequence data were analyzed using highly conserved, orthologous mapk activation loop sequences. the activation of map kinases occurs by a dual phosphorylation event by the upstream kinase on a solvent-exposed activation loop that typically contains a t-x-y motif. we used the encoded activation loop sequences from d. melanogaster slipper and d. melanogaster wallenda, flanked by 5- additional conserved amino residues, in tblastn queries against an. gambiae sequence data. conserved catalytic domains for all predicted proteins were confirmed using prosite. phylogenetic analysis for phylogenetic analyses, sequences, ranging from 115- amino acids, of mapk catalytic domains from an. gambiae, c. elegans, ciona intestinalis, d. melanogaster, homo sapiens and saccharomyces cerevisiae were used to generate an alignment using the blosum protein weight matrix and clustalx. orthologs of c. elegans, d. melanogaster and c. intestinalis mapks were confirmed using blastp of genecards, ensemble metazoa, wormbase and flybase databases. phylogenetic analyses were conducted using phylogeny inference package. protdist was used to generate a distance matrix based on the jones-taylor-thornton model of amino acid substitution. a distance-based phylogenetic tree was then inferred using the neighbor-joining algorithm implemented in neighbor. seqboot was used to prepare pseudoreplicate datasets for bootstrap analysis; these replicate datasets were then analyzed using protdist with the jtt model and neighbor to create a bootstrap nj tree. the bootstrap majority-rule consensus tree was generated using consense, visualized in figtree v. http://tree.bio.ed.ac.uk/software/figtree/ and edited using inkscape http://inkscape.org/. the unrooted consensus tree was drawn to scale, with branch lengths representing the mean number of substitutions per site. mosquito cell culture and experimental treatments the immortalized, embryo-derived an. gambiae 4a3b cell line was maintained in schneider's medium with 10% heat-inactivated fetal bovine serum at 28°c. for each condition, × 4a3b cells in ml medium were plated in one well of a 12-well tissue culture plate and allowed to recover overnight. cells were then treated with the following stimuli at concentrations previously validated for signaling: μm hydrogen peroxide, pg/ml human tgf-beta, μm human insulin or μg/ml lipopolysaccharide and collected at min, min, min, h, h, h and h after treatment for subsequent western blot analysis. to quantify and analyze erk, jnk, and p mapk transcript expression, 4a3b cells prepared as described above were treated with μm human insulin and collected at h, h, h and h for qpcr. controls for each time point consisted of an identical aliquot of cells treated with diluent. at least three biological replicates were performed for each treatment. protein extraction and western blotting protein extracts of 4a3b cells were prepared by collecting cells in lysis buffer as previously described. briefly, cell medium was removed following treatment and cells were washed with ice cold phosphate buffered saline and lysed in the plate in μl cell lysis buffer. the plate was agitated for min at 4°c and samples were incubated on ice for min. cell lysates were centrifuged at × g for min at 4°c to remove cellular debris and μl of supernatant from each sample was mixed with μl of × sample buffer and heated at 95°c for min. protein samples were electrophoretically separated on 10% polyacrylamide gels via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. protein loading was visually assessed by coomassie blue staining. membranes were blocked in 5% nonfat dry milk in × tris-buffered saline containing % tween- for h at room temperature. for phosphorylated erk detection, membranes were incubated at 4°c overnight with 1: mouse anti-phospho-erk monoclonal antibody in 5% nfdm in tbs-t. for detection of phospho-p phospho-jnk and the loading control protein glyceraldehyde 3-phosphate dehydrogenase, membranes were incubated at 4°c overnight with 1: rabbit anti-phospho-p mapk antibody, 1: rabbit anti-phospho-jnk1/ antibody, or with 1: rabbit anti-gapdh antibody in 5% nfdm in tbs-t. membranes were washed times for min in × tbs-t and incubated with a 1: dilution of horse radish peroxidase -conjugated rabbit anti-mouse igg or with a 1: dilution of hrp-conjugated goat anti-rabbit fragment at 4°c overnight. following incubation with the secondary antibody, membranes were washed times for min in × tbs-t. to reveal antibody-bound proteins, membranes were incubated with supersignal west pico chemiluminescent reagent for 2- min. each membrane was exposed to blue autoradiography film. phospho-mapk levels and total gapdh levels were quantified on scanned film using a gs- calibrated densitometer. mapk quantitative pcr primers for qpcr of an. gambiae erk, p jnka, and jnkb were designed using primer3plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi. the primers included: erk, 5'atcccgagcacgatcaca3' and 5'cgattttgtgtagcccttgga3'; p mapk, 5'cggaccacattcaccagcta3' and 5'cgctaaagttgcgcttctcc3'; jnka, 5'gcacgcagcgatacattagc3' and 5'gtccaccgaaatcctttcca3'; and jnkb, 5'gaccgactcgaacgagcac3' and 5'cgaaccacacgttgatgtagc3'. the housekeeping gene encoding ribosomal protein s was analyzed as a control with the primers 5'gaaggccttccagaaggtacaga3' and 5'catcggtttgggcagaatg3'. rna was isolated from 4a3b cells using trizol regent and contaminating dna was removed from the rna samples using turbo dna-free™. all rna samples were diluted to ng/μl and μl rna from each sample was reverse-transcribed using superscript® iii. sample cdnas were used to perform qpcr using maxima sybr green/rox qpcr master mix. the qpcr cycling conditions were as follows: min at 50°c, min at 95°c, and cycles of s at 95°c and min at 60°c. three biological replicates with independent batches of 4a3b cells were analyzed for the diluent controls and treatments. no template controls were included for each reaction plate and all reactions were performed in triplicate to confirm amplification consistency. the triplicate data were used to determine an average ct for each reaction and the data analyzed using the 2-ΔΔct method as described. briefly, the average ct value for each target was first normalized to the average ct for ribosomal protein s gene to obtain Δct, and then the Δct value for each insulin-treated sample was normalized to timepoint-matched control to obtain ΔΔct. fold-change of expression relative to the diluent control was determined using 2-ΔΔct. statistical analyses for western blotting data, the kolmogorov-smirnov test revealed that non-normal distributions were present in our data sets. overall significance values of all data sets, therefore, were determined by the kruskal-wallis test and dunn's post test for pairwise comparisons. for the q-pcr data, one way anova and student-neuman-keuls were used for analyses of significance.  mapk discovery we used a combination of bioinformatic techniques to identify a total of mapks from the an. gambiae genome. among these, agap and agap were newly identified mapk orthologs. based on homology, agap is most appropriately defined as an. gambiae map3k10/ or mixed lineage kinase 2/. similarly, agap is most appropriately defined as an. gambiae map3k 12/. agap and agap are annotated as p mapk and jnk orthologs, respectively, in vectorbase. however, inspection of agap by alignment with p mapk orthologs and reverse psi -blast for conserved domains revealed that the annotated sequence was missing key n-terminal residues in the kinase catalytic domain. specifically, rps-blast of the annotated sequence predicted only of active site and of activation loop residues. because the annotation appeared to be incomplete, we used the first encoded amino acids from the n-terminus of the ae. aegypti p mapk ortholog to query the national center for biotechnology information an. gambiae trace archives database and identified the probable n-terminus of agap. similarly, the annotation of agap in vectorbase does not encode a start methionine. approximately base pairs of sequence upstream of the 5'-most nucleotides of agap were translated in the three forward reading frames and analyzed by blastx using the first amino acids of the corresponding orthologs from ae. aegypti and from culex quinquefasciatus. a putative start methionine with kozak consensus and additional n-terminal amino acids extended the encoded sequence of agap. the map4ks, the map3ks, the map2ks are present in 1: orthology with gene products that have been identified in d. melanogaster. among the mapks, only d. melanogaster rolled and an. gambiae erk are in 1: orthology. in contrast, the d. melanogaster genome encodes two p mapk orthologs, a single jnk ortholog, an erk ortholog, and an erk ortholog, although the latter two are not associated with prototypical map kinase cascades. the an. gambiae genome, on the other hand, encodes a single p mapk, two jnk orthologs, and no erk and erk orthologs. based on sequence homology, an. gambiae jnkb is most closely related to d. melanogaster basket. the lack of strict 1: orthology among the mapks is perhaps not surprising because diversification among these proteins to accommodate unique aspects of host biology would have a greater likelihood of being tolerated relative to upstream changes in the signaling cascades that could result in significantly amplified non-adaptive phenotypes. to determine whether the complement of mapks in the an. gambiae genome was similar to that in other mosquito genomes, we examined the available erk, jnk, and p mapk orthologs for ae. aegypti and cx. quinquefasciatus. the genome sequences of ae. aegypti and cx. quinquefasciatus are currently assembled into supercontigs in vectorbase. in this context, if identical nucleotide sequences are excluded, erk, jnka, and jnkb orthologs are in 1:1: orthology among the three mosquito species. in contrast, cx. quinquefasciatus appears to possess two relatively short mapks that are tentatively identified as p mapks. the encoded sequences share significant identity to ae. aegypti p mapk and the putative full-length an. gambiae p mapk, but are non-overlapping sequences, so it is impossible to discern whether these are indeed truly distinct gene products encoded in the cx. quinquefasciatus genome. because these relationships cannot be confirmed with the existing genome assemblies, the ae. aegypti and cx. quinquefasciatus mapk sequences were excluded from the phylogenetic analysis. phylogeny phylogenetic analysis of the mapk catalytic domains showed strong support for the map4k and map2k groups. the map3k raf superfamily formed a group, but the map3k mekk family did not form an exclusive group. similarly, the two map3k superfamilies did not group together as an exclusive group and the sequences representing mapk were not supported as a group in this analysis. the lack of a close relationship between the raf and mekk map3k groups was not unexpected based on previous phylogenetic analyses. the mekk family proteins and the raf superfamily proteins are both associated with p and jnk signaling pathways; however, raf proteins also figure prominently in mek/erk signaling. although not reflected in this analysis, the previously noted division of the erk subfamily may be due to the classification of erk and 7/ as mapk-like cascade independent kinases. frequently, sequences from the two deuterostomes and the insects grouped together. exceptions to these groupings occurred when there were no clear homologs, which likely reflects the sequence diversification that has resulted in the many mapks that are found in humans relative to other species. functional assays the identification of the complement of map4ks, map3ks, map2ks and mapks provides a framework for the prediction of patterns of activation of cell signaling cascades in an. gambiae cells. to attempt to determine whether these pathways are phenotypically represented in this mosquito species, we selected three stimuli that we had previously implicated in erk- and p mapk-dependent an. gambiae immune signaling, as well as lipopolysaccharide, an activator of immune signaling pathways in mosquito cells, to stimulate an. gambiae 4a3b cells over an extended timecourse from min to h. a representative western blot is shown in additional file stimulation with μm hydrogen peroxide induced significant activation of p mapk at min relative to the control group. signaling declined from min to h post-stimulation, with a return to control levels by h. despite trends toward induction within h of stimulation, erk and jnk were not significantly activated by μm hydrogen peroxide in 4a3b cells. in previous studies, stimulation of 4a3b cells with μm hydrogen peroxide induced activation of p mapk as well as erk by min post-treatment, a faster and more extensive mapk induction than observed here for μm hydrogen peroxide. in accord with previous observations at min post-treatment, stimulation with μm human insulin induced significant activation of erk and p mapk at min relative to the control and min time point. in contrast to the more temporally limited activation by hydrogen peroxide, insulin-activated erk and p mapk were detectable to h and h post-stimulation, respectively, suggesting a greater persistence of these signaling responses. phosphorylation of jnk was slightly, although not significantly, elevated relative to control levels within h after stimulation. stimulation with pg/ml human tgf-beta induced significant activation of erk only at min post-treatment relative to the min time point, with detectable levels of activated erk through h post-stimulation. this induction of erk activation at min, together with a lack of p mapk activation, was noted previously. in contrast to slight trends toward jnk activation by hydrogen peroxide and insulin, phosphorylated jnk levels never exceeded baseline control levels at all times after stimulation with human tgf-beta. stimulation with μg/ml lps presented a unique pattern of mapk activation in an. gambiae 4a3b cells. in contrast to erk and p mapk activation by hydrogen peroxide, insulin and tgf-beta, lps stimulation did not induce mean phosphorylated erk levels above baseline and only non-significant trends toward elevated phosphorylated p mapk were noted through min post-stimulation. levels of phosphorylated jnk, however, were significantly increased by lps stimulation at min and min post-treatment relative to control, with slightly elevated but not significant levels of phosphorylated jnk through h post-treatment that declined to baseline control levels by h post-treatment. although the majority of signaling protein genes do not exhibit transcriptional responsiveness to environmental stimuli, we questioned whether we might detect a signaling response at the mapk transcript level in an. gambiae cells. to address this question, we used qpcr to analyze expression levels of p mapk, jnka, jnkb, and erk expression in response to human insulin, the only stimulus for which we detected changes in phosphorylation for more than one mapk. in replicated assays, none of the four genes exhibited a significant change in expression from h to h after treatment with μm human insulin. together with our phosphorylation data, these data suggest that mapk responsiveness in an. gambiae cells occurs principally at the protein level. discussion the protein targets of the mammalian mapks predict a strong association of the mapks with immune regulation. this association is also ancient, with strong representation in invertebrates. in c. elegans, p mapk- and perk-dependent signaling pathways, as well as insulin and tgf-beta signaling pathways, are required for host defense, with the general presumptions that insulin signaling functions in pathogen surveillance, p mapk-dependent signaling functions in general defense, and p mapk-independent signaling pathways provide pathogen-specific responses. this nematode, however, lacks orthologs of rel and nuclear factor -kappab, the primary immune signaling transcription factors of d. melanogaster. in the fruit fly, toll- or imd-dependent signaling regulates nf-kappab and jnk pathways, while p mapk-dependent signaling mediates host defense against pathogen invasion. the jnk and p mapks also mediate control of antimicrobial peptide expression in d. melanogaster, but cooperativity with nf-κb appears to be restricted to jnk. in particular, imd activation results in the activation of tak upstream of jnk and i-kappa kinase, the upstream activator of rel that ultimately feeds back to reduce jnk activation. the complement of map kinases encoded in the an. gambiae genome would provide this organism with the significant redundancy of p mapk and jnk signaling observed in model organisms, although some intriguing differences between the mosquito and other species are predicted as well. in particular, an. gambiae map4k may function similarly to its mammalian counterpart downstream of toll and rac to activate the mosquito mlk2/ ortholog and p mapk and jnk signaling. the second an. gambiae map4k - map4k - would be predicted to activate map3k and map3k however, an. gambiae lacks an ortholog for map3k an upstream activator for mek/erk signaling, so map4k activation of map3k would be predicted to activate nf-kappab signaling through ikk, and p mapk and jnk signaling through map2k and map2k activation, respectively. among the other an. gambiae map3k orthologs, a number are predicted to lack well-defined upstream activators or to function independently of map4k activation. in particular, the activators of an. gambiae ortholog of map3k12/ are not well defined based on data from mammalian models; nevertheless, the mosquito protein would be predicted to contribute to the activation of jnk signaling. among the map3k orthologs that can be activated independently of map4ks, an. gambiae map3k would be predicted to lie downstream of tgf-beta activation of gadd, while map3k could be activated by tgf-beta-mediated activation of daxx or tnf/tnfr activation of traf. eiger and wengen have been identified as d. melanogaster orthologs of tnf/tnfr and, in the context of innate immunity, eiger functions independently of the toll and imd signaling pathways to regulate pathogen-induced cell death, proliferation of extracellular pathogens and prophenoloxidase-mediated defense responses. although a clear ortholog of traf is not apparent in the an. gambiae genome, orthologs of eiger and wengen would suggest that signaling by this ligand and receptor could lead to map3k5/map2k activation of p mapk. an examination of possible pathways leading to mek/erk signaling based on the encoded map kinases reveals that, in addition to a lack of map3k an. gambiae also lacks an ortholog for map3k so the regulation of mek/erk signaling is likely dependent on b-raf and mos activation, which are the downstream targets for rap and for ras, a major transducer for multiple signaling inputs from growth factors and cytokines. the data from our functional studies not only confirm a subset of signaling predictions from known pathways, but also suggest that other pathways may be involved in an. gambiae mapk activation. in particular, human insulin-induced activation of an. gambiae erk was predicted and this was consistent with previous observations. in contrast, activation of an. gambiae p mapk by insulin, which may occur through hydrogen peroxide at a concentration that is insufficient for erk activation, is not predicted to flow through map4k-map3k-map2k activation. interestingly, several studies have reported cross talk between the insulin receptor kinase and the janus-activated kinase signaling pathways. in particular, jak - the major mediator of inflammatory cytokine signaling in mammalian cells - can be activated by hydrogen peroxide and contributes to insulin-dependent activation of p mapk, jnk, and erk in a manner that is independent of insulin receptor substrate activation and activation of the phosphatidylinositol 3-kinase/akt-dependent arm of insulin signaling. jaramillo-gutierrez et al. proposed, based on the regulation of antioxidant gene expression by jnk, that this mapk may in fact be induced by oxidative stress to control redox biology in an. gambiae. these observations - together with our data and known models of mapk signaling - predict the existence of a pathway involving insulin-induced hydrogen peroxide regulation of a jak ortholog and downstream activation of p mapk and jnk that can be tested experimentally in an. gambiae. in contrast to the prediction of erk-specific signaling by insulin, existing mammalian signaling data predict that human tgf-beta should activate erk as well as p mapk in an. gambiae cells. we have extensively characterized tgf-beta1-dependent erk signaling in the regulation of p. falciparum development in an. stephensi and we have also demonstrated that p mapk is activated by tgf-beta in an. stephensi cells. given that p mapk can be activated in an. gambiae cells, our understanding of mammalian mapk signaling would suggest that perhaps a deficiency in the tgfbr-gadd45-map3k cascade is responsible for the failure of tgf-beta to activate p mapk in an. gambiae 4a3b cells. a comparative analysis of an. stephensi and an. gambiae 4a3b cells, therefore, could be used to identify critical differences in these signaling pathways. lps has been used as an activator of a variety of immune signaling pathways in mosquito cells. a putative an. gambiae rac1-map4k1-map3k11-map2k7-jnk signaling cascade could provide one mechanism for lps activation of jnk in an. gambiae 4a3b cells. however, we could also predict, given that this mosquito map3k is orthologous with map3k as well as map3k that lps should also activate p mapk signaling through map2k because lps does not significantly activate p mapk in an. gambiae 4a3b cells, we could speculate that this an. gambiae map3k is functionally more analogous to map3k an examination of map2k and map2k activation downstream of an. gambiae map3k10/ using inhibitors or knock down strategies could clarify the orthology and functionality of this mosquito map3k. although lps was the only pathogen-associated molecular pattern used in these signaling studies, the restriction of mapk activation by lps to an. gambiae jnk is insightful. akman-anderson et al. showed that p. falciparum hemozoin induced activation of tak and erk as well as the signaling kinases akt/protein kinase b and atypical protein kinase c zeta/lambda in an. gambiae 4a3b cells. activation of tak by this important parasite signaling factor, together with lps-specific activation of jnk in our studies, suggests that jnk may function specifically in pathogen detection in an. gambiae cells. indeed, following infection with p. berghei or p. falciparum, the mosquito midgut epithelium undergoes profound cytoskeletal changes similar to those regulated by the toll/map2k4/jnk signaling module in d. melanogaster. additional support of the importance, and perhaps specificity, of jnk signaling for control of natural p. falciparum infection in an. gambiae is provided by two population genetics studies. we identified a single nucleotide polymorphism in the an. gambiae map2k gene that was in linkage disequilibrium with a snp in the gene encoding toll5b that was significantly associated with p. falciparum infection. this synonymous snp, mkk introduces a reduction of codon frequency greater than 3-fold, which could result in changes in downstream protein expression or function and, hence, jnk signaling. in addition to our work, riehle et al. mapped a locus that is strongly associated with an. gambiae resistance to p. falciparum infection in east africa that encompasses the chromosomal locations of map4k and map3k both of which are predicted to regulate jnk signaling.  in sum, we have identified the complement of an. gambiae map kinases and used a combination of predicted signaling relationships and bioassays to suggest novel interactions and functionality that can be tested experimentally in this biomedically important mosquito species. clearly, the mapk signaling cascades are the major regulators of innate immunity in c. elegans, in d. melanogaster, and in mammals, but we are only beginning to appreciate the importance of these complex cascades in innate immunity of the mosquito genera that serve as vectors of globally devastating pathogens of humans. the establishment of this "road map" based on the most advanced mosquito genome annotation can, therefore, accelerate our understanding of host-pathogen interactions and broader physiology in an. gambiae and in the closely related an. stephensi and provide a reasonable architecture for similar efforts in ae. aegypti and cx. quinquefasciatus. further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the map kinase cascades and other networked pathways, will ultimately be necessary to elucidate the "master signaling regulators" in these biomedically important insects. list of abbreviations ask: apoptosis signal-regulating kinase; blastx: basic local alignment search tool for translated query against a protein database; daxx: death-associated protein 6; dlk: dual leucine zipper kinase; erk: extracellular signal-regulated kinase; gadd45: growth arrest and dna-damage inducible protein; gapdh: glyceraldehyde 3-phosphate dehydrogenase; hrp: horse radish peroxidase; ikk: i kappa b kinase; jak-stat: janus kinase/signal transducers and activators of transcription; jnk: jun kinase; lps: lipopolysaccharide; map4k: mitogen-activated protein kinase kinase kinase kinase; map3k: mitogen-activated protein kinase kinase kinase; map2k: mitogen-activated protein kinase kinase; mapk: mitogen-activated protein kinase; mek: equivalent to map2k; mekk: equivalent to map3k; mlk: mixed lineage kinase; nfdm: nonfat dry milk; nf-kappab: nuclear factor-kappab; no: nitric oxide; pfgpis: plasmodium falciparum glycosylphosphatidylinositols; prke1: equivalent to map3k mlk1; rac1: ras-related c botulinum toxin substrate 1; rap1: ras-related protein 1; tak1: tgf-beta-activated kinase 1; tblastn: basic local alignment search tool for protein query against a translated database; tbs-t: × tris-buffered saline with % tween 20; tgf-beta1: transforming growth factor-beta1; tnfr: tumor necrosis factor receptor; traf2:/6: tnfr-associated factor 2/ authors' contributions aah and msp confirmed the identities of the annotated and unannotated an. gambiae mapks, co-wrote the initial draft, and assisted in the preparation of figures/files. aah also edited the manuscript and revisions. bw performed the mapk signaling and expression assays with an. gambiae cells, analyzed the data, and edited the manuscript and revisions. lc and san performed the phylogenetic analyses, prepared and edited the phylogram, and edited the manuscript. aa, mn and jrf created the mapk signaling diagram based on the kegg pathway database and current literature and edited the manuscript. sl conceived of this work, directed the studies, and co-wrote the initial and final drafts for publication. all authors read and approved the final manuscript. supplementary material additional file 1 mapks used in this study. h. sapiens, d. melanogaster, c. elegans and c. intestinalis are listed by common names and by accession number. an. gambiae mapks are listed by vectorbase agap numbers as well as by ncbi accession numbers and common names. dashes indicate the absence of identified orthologs. n/a for an. gambiae map3k indicates no available ncbi accession number. click here for file additional file 2 alignment of predicted mosquito p mapks. alignment of an. gambiae, ae. aegypti and cx. quinquefasciatus p mapks. the ae. aegypti and cx. quinquefasciatus sequences were used to query the an. gambiae trace archives database to identify the probable n-terminal amino acids of an. gambiae p mapk. click here for file additional file 3 alignment of an. gambiae jnka and ae. aegypti and cx. quinquefasciatus jnk orthologs. alignment of the first amino acids of ae. aegypti and cx. quinquefasciatus jnk orthologs with upstream sequence from agap was used to predict the start methionine and additional n-terminal amino acids of an. gambiae jnka. click here for file additional file 4 predicted amino acid sequences of erk, jnk, p mapk orthologs from ae. aegypti and cx. quinquefasciatus. ae. aegypti and cx. quinquefasciatus mapk orthologs as predicted from the supercontig assemblies for genome sequences for these species. these sequences were excluded from the phylogeny and, therefore, the relationships of these orthologs with an. gambiae mapks cannot definitively be confirmed. click here for file additional file 5 lps-induced mapk phosphorylation in an. gambiae 4a3b cells. cells were treated with μg/ml lps or an equivalent volume of pbs. cells were collected at min, min, h, h, h and h after treatment. mapk phosphorylation was examined by western blotting as described in the methods. gapdh levels provided an assessment of protein loading and were used to normalize corresponding phospho-mapk levels. this figure is a representative of immunoblots from 6- independent experiments. click here for file additional file 6 mapk transcript expression in insulin-stimulated an. gambiae 4a3b cells. cells were treated with μm human insulin or an equivalent volume of diluent and collected at h, h, h and h after treatment. expression levels of erk, p mapk, jnka, and jnkb were analyzed by qpcr as described in the methods. expression of ribosomal protein s a housekeeping gene control, was used for normalization of treatment and control expression. insulin-treated mapk expression levels are shown as fold changes relative to the timepoint-matched control group levels. click here for file acknowledgements and funding we acknowledge support provided by hannah smithers, kong wai cheung and andrew ross for technical support for cell culture experiments and dr. ian korf for computational support. this work was funded by the national institutes of health national institute of allergy and infectious diseases r ai and r ai to sl.
further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the map kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species. the establishment of a mapk "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of an. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection and to bacterial lipopolysaccharide. gambiae mapks, two of which were previously unknown and two others that were incompletely annotated. our understanding of the roles of the mapk signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted. however, in addition to nf-kappab-dependent signaling, three mitogen-activated protein kinase pathways regulated by jnk, erk and p mapk are critical mediators of innate immunity in other invertebrates and in mammals. anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-saharan africa. to date, three innate immune signaling pathways, including the nuclear factor -kappab-dependent toll and immune deficient pathways and the janus kinase/signal transducers and activators of transcription pathway, have been extensively characterized in an.
 anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-saharan africa. to date, three innate immune signaling pathways, including the nuclear factor -kappab-dependent toll and immune deficient pathways and the janus kinase/signal transducers and activators of transcription pathway, have been extensively characterized in an. gambiae. however, in addition to nf-kappab-dependent signaling, three mitogen-activated protein kinase pathways regulated by jnk, erk and p mapk are critical mediators of innate immunity in other invertebrates and in mammals. our understanding of the roles of the mapk signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.  in this study, we present the orthologs and phylogeny of an. gambiae mapks, two of which were previously unknown and two others that were incompletely annotated. we also provide detailed temporal activation profiles for erk, jnk, and p mapk in an. gambiae cells in vitro to immune signals that are relevant to malaria parasite infection and to bacterial lipopolysaccharide. these activation profiles and possible upstream regulatory pathways are interpreted in light of known mapk signaling cascades.  the establishment of a mapk "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of an. gambiae and other mosquito species. further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the map kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species. 