In Utero Heat Stress Alters the Offspring Epigenome.

Exposure to intrauterine heat stress during late gestation affects offspring performance into adulthood. However, underlying mechanistic links between thermal insult in fetal life and postnatal outcomes are not completely understood. We examined morphology, DNA methylation, and gene expression of liver and mammary gland for bull calves and heifers that were gestated under maternal conditions of heat stress or cooling (i.e. in utero heat stressed vs. in utero cooled calves). Mammary tissue was harvested from dairy heifers during their first lactation and liver from bull calves at birth. The liver of in utero heat stressed bull calves contained more cells and the mammary glands of in utero heat stressed heifers were comprised of smaller alveoli. We identified more than 1,500 CpG sites differently methylated between maternal treatment groups. These CpGs were associated with approximately 400 genes, which play a role in processes, such as development, innate immune defense, cell signaling, and transcription and translation. We also identified over 100 differentially expressed genes in the mammary gland with similar functions. Interestingly, fifty differentially methylated genes were shared by both bull calf liver and heifer mammary gland. Intrauterine heat stress alters the methylation profile of liver and mammary DNA and programs their morphology in postnatal life, which may contribute to the poorer performance of in utero heat stressed calves.

Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton.

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development, as well as their biotic and abiotic stress responses. As a nodal point of the MAPK cascade, the MKK gene family has not been systematically studied in cotton. Here, we identified 11 putative MKK genes in the Gossypium raimondii genome. Phylogenetic analysis showed that the MKKs were supported by architectures of conserved protein motifs. Expression patterns of MKKs under hormone treatments or abiotic stresses revealed their diverse functions in stress responses. Based on a yeast two hybrid, a total of 63 interactive pairs of MKKs and MAPKs were identified in cotton. Among these, 40 interactive pairs were newly identified compared to that reported previously in Arabidopsis. Integration analysis of the interaction network and expression patterns of MKK and MAPK family members revealed 13 potential MAPK signaling modules that are involved in the complicated cross-talk between hormones and abiotic stresses. Taken together, our data enhance the understanding of the evolution and function of MAPK cascades in cotton, and lay the foundation for the improvement of various defense responses that use MAPK signaling modules in the future.

Genome-Wide Identification of Mitogen-Activated Protein Kinase Gene Family across Fungal Lineage Shows Presence of Novel and Diverse Activation Loop Motifs.

The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest.

Cross-Family Comparative Proteomic Study and Molecular Phylogeny of MAP Kinases in Plants.

Mitogen-activated protein kinases are serine/threonine-specific protein kinases and they are closely related to cyclin-dependent kinases. They constitute functionally significant family of proteins that is involved in various cellular functions like response to mitogens, osmotic stress, heat shock and proinflammatory cytokines as well as known to play key role in proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis. MAP kinases are characteristically found in eukaryotes only, though they are fairly diverse and encountered in all animals, fungi and plants, and even in an array of unicellular eukaryotes. In this study 24 MAP kinase sequences from various plant species were selected in order to compare their conserved regions, amino acid composition, evolutionary orders and other statistical parameters.

Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon.

BACKGROUND: Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood. RESULTS: In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study. CONCLUSIONS: BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.

A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a ß-1,3-glucan synthase, decreased chitin content, 'budded' hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading.

Nemo-like kinase, a multifaceted cell signaling regulator.

Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-related kinase. Although NLK was originally identified as a Drosophila gene affecting cell movement during eye development, recent studies show that NLK also contributes to cell proliferation, differentiation, and morphological changes during early embryogenesis and nervous system development in vertebrates. In addition, NLK has been reported to be involved in the development of several human cancers. NLK is able to play a role in multiple processes due to its capacity to regulate a diverse array of signaling pathways, including the Wnt/ß-catenin, Activin, IL-6, and Notch signaling pathways. Although the molecular mechanisms that regulate NLK activity remain unclear, our recent research has presented a new model for NLK activation. Here, we summarize the current understanding of the function and regulation of NLK and discuss the aspects of NLK regulation that remain to be resolved.

Induction of MAP kinase homologues during growth and morphogenetic development of Karnal bunt (Tilletia indica) under the influence of host factor(s) from wheat spikes.

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) in response to certain environmental conditions, play important role in mating type switching (Fus3) and pathogenicity (Pmk1) in many fungi. In order to determine the roles of such regulatory genes in Tilletia indica, the causal pathogen of Karnal bunt (KB) of wheat, semi-quantitative and quantitative RT-PCR was carried out to isolate and determine the expression of MAP kinase homologues during fungal growth and development under in vitro culture. Maximum expression of TiFus3 and TiPmk1 genes were observed at 14th and 21st days of culture and decreased thereafter. To investigate whether the fungus alters the expression levels of same kinases upon interaction with plants, cultures were treated with 1% of host factors (extracted from S-2 stage of wheat spikes). Such treatment induced the expression of MAPks in time dependent manner compared to the absence of host factors. These results suggest that host factor(s) provide certain signal(s) which activate TiFus3 and TiPmk1 during morphogenetic development of T. indica. The results also provides a clue about the role of host factors in enhancing the disease potential due to induction of MAP kinases involved in fungal development and pathogenecity.

Mitogen-activated protein kinase signaling in plant-interacting fungi: distinct messages from conserved messengers.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved proteins that function as key signal transduction components in fungi, plants, and mammals. During interaction between phytopathogenic fungi and plants, fungal MAPKs help to promote mechanical and/or enzymatic penetration of host tissues, while plant MAPKs are required for activation of plant immunity. However, new insights suggest that MAPK cascades in both organisms do not operate independently but that they mutually contribute to a highly interconnected molecular dialogue between the plant and the fungus. As a result, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant signaling, including the recruitment of plant MAPK cascades. Conversely, plant MAPKs promote defense mechanisms that threaten the survival of fungal cells, leading to a stress response mediated in part by fungal MAPK cascades. In this review, we make use of the genomic data available following completion of whole-genome sequencing projects to analyze the structure of MAPK protein families in 24 fungal taxa, including both plant pathogens and mycorrhizal symbionts. Based on conserved patterns of sequence diversification, we also propose the adoption of a unified fungal MAPK nomenclature derived from that established for the model species Saccharomyces cerevisiae. Finally, we summarize current knowledge of the functions of MAPK cascades in phytopathogenic fungi and highlight the central role played by MAPK signaling during the molecular dialogue between plants and invading fungal pathogens.

Molecular characterization of a Fus3/Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1.

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes the destructive wheat stripe rust disease worldwide. Due to the lack of reliable transformation and gene disruption method, knowledge about the function of Pst genes involved in pathogenesis is limited. Mitogen-activated protein kinase (MAPK) genes have been shown in a number of plant pathogenic fungi to play critical roles in regulating various infection processes. In the present study, we identified and characterized the first MAPK gene PsMAPK1 in Pst. Phylogenetic analysis indicated that PsMAPK1 is a YERK1 MAP kinase belonging to the Fus3/Kss1 class. Single nucleotide polymerphisms (SNPs) and insertion/deletion were detected in the coding region of PsMAPK1 among six Pst isolates. Real-time RT-PCR analyses revealed that PsMAPK1 expression was induced at early infection stages and peaked during haustorium formation. When expressed in Fusarium graminearum, PsMAPK1 partially rescued the map1 mutant in vegetative growth and pathogenicity. It also partially complemented the defects of the Magnaporthe oryzae pmk1 mutant in appressorium formation and plant infection. These results suggest that F. graminearum and M. oryzae can be used as surrogate systems for functional analysis of well-conserved Pst genes and PsMAPK1 may play a role in the regulation of plant penetration and infectious growth in Pst.

GhMPK7, a novel multiple stress-responsive cotton group C MAPK gene, has a role in broad spectrum disease resistance and plant development.

Mitogen-activated protein kinase (MAPK) cascades play a pivotal role in environmental responses and developmental processes in plants. Previous researches mainly focus on the MAPKs in groups A and B, and little is known on group C. In this study, we isolated and characterized GhMPK7, which is a novel gene from cotton belonging to the group C MAPK. RNA blot analysis indicated that GhMPK7 transcript was induced by pathogen infection and multiple defense-related signal molecules. Transgenic Nicotina benthamiana overexpressing GhMPK7 displayed significant resistance to fungus Colletotrichum nicotianae and virus PVY, and the transcript levels of SA pathway genes were more rapidly and strongly induced. Furthermore, the transgenic N. benthamiana showed reduced ROS-mediated injuries by upregulating expression of oxidative stress-related genes. Interestingly, the transgenic plants germinated earlier and grew faster in comparison to wild-type plants. beta-glucuronidase activity driven by the GhMPK7 promoter was detected in the apical meristem at the vegetative stage, and it was enhanced by treatments with signal molecules and phytohormones. These results suggest that GhMPK7 might play an important role in SA-regulated broad-spectrum resistance to pathogen infection, and that it is also involved in regulation of plant growth and development.

Accelerated evolution of PAK3- and PIM1-like kinase gene families in the zebra finch, Taeniopygia guttata.

Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain.

In vivo functions of mitogen-activated protein kinases: conclusions from knock-in and knock-out mice.

Multicellular organisms achieve intercellular communication by means of signalling molecules whose effect on the target cell is mediated by signal transduction pathways. Such pathways relay, amplify and integrate signals to elicit appropriate biological responses. Protein kinases form crucial intermediate components of numerous signalling pathways. One group of protein kinases, the mitogen-activated protein kinases (MAP kinases) are kinases involved in signalling pathways that respond primarily to mitogens and stress stimuli. In vitro studies revealed that the MAP kinases are implicated in several cellular processes, including cell division, differentiation, cell survival/apoptosis, gene expression, motility and metabolism. As such, dysfunction of specific MAP kinases is associated with diseases such as cancer and immunological disorders. However, the genuine in vivo functions of many MAP kinases remain elusive. Genetically modified mouse models deficient in a specific MAP kinase or expressing a constitutive active or a dominant negative variant of a particular MAP kinase offer valuable tools for elucidating the biological role of these protein kinases. In this review, we focus on the current status of MAP kinase knock-in and knock-out mouse models and their phenotypes. Moreover, examples of the application of MAP kinase transgenic mice for validating therapeutic properties of specific MAP kinase inhibitors, and for investigating the role of MAP kinase in pathogen-host interactions will be discussed.

Statistics for proteomics: a review of tools for analyzing experimental data.

Most proteomics experiments make use of 'high throughput' technologies such as 2-DE, MS or protein arrays to measure simultaneously the expression levels of thousands of proteins. Such experiments yield large, high-dimensional data sets which usually reflect not only the biological but also technical and experimental factors. Statistical tools are essential for evaluating these data and preventing false conclusions. Here, an overview is given of some typical statistical tools for proteomics experiments. In particular, we present methods for data preprocessing (e.g. calibration, missing values estimation and outlier detection), comparison of protein expression in different groups (e.g. detection of differentially expressed proteins or classification of new observations) as well as the detection of dependencies between proteins (e.g. protein clusters or networks). We also discuss questions of sample size planning for some of these methods.

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily.

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.

Isolation and identification of SA and JA inducible protein kinase gene OsSJMK1 in rice.

On the basis of a JA-inducible EST, the full length cDNA of a putative protein kinase gene, OsSJMKJ, was isolated from rice (Oryza sativa L.). The predicted protein of this gene, consisting of 498 amino acids with a predicted PI value of 8.43, contains a mitogen-activated protein kinase (MAPK) domain with a dual-phosphorylation activation motif TDY (Thr-Asp-Tyr) and a C-terminus domain that feature a typical MAPK belonging to subgroup V. OsSJMK1 showed 81% sequence identity to the reported BWMK1, a blast fungus and wounding inducible protein, in the N-terminus kinase domain but had low sequence identity in the C-terminus domain. Despite the high similarity of gene structure between OsSJMK1 and BWMK1, the expression of OsSJMK1 was weakly induced by blast fungus but not wounding, rather it was transiently induced by jasmonic acid (JA) and salicylic acid (SA) at early stages of treatment. This gene was not induced by other hormones (such as ABA) or abiotic stresses, such as drought and salinity. The low level of transcript of this gene was detected in the various tissues of rice. The results suggest that OsSJMK1 might be activated specifically by JA and SA and involved in defense signaling pathways.

Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection.

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. In Arabidopsis, 20 MAPKs have been identified and divided into four major groups. In rice, a monocot model and economically important cereal crop, only five MAPKs were characterized, including three related to the host defense response. In this study, we have identified 17 members of the rice MAPK gene (OsMPK) family through an in silico search of rice genome databases. Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, we propose that MAPKs can be divided into six groups. Interestingly, the rice genome contains many more MAPKs with the TDY phosphorylation site (11 members) than with the TEY motif (six members). In contrast, the Arabidopsis genome contains more MAPKs with the TEY motif (12 members) than with the TDY motif (eight members). Upon inoculation with the blast fungus (Magnaporthe grisea), nine of 17 OsMPK genes were found to be induced at the mRNA level during either early, late, or both stages of infection. Four of the M. grisea-induced OsMPK genes were associated with host-cell death in the lesion-mimic rice mutant, and eight of them were differentially induced in response to defense signal molecules such as jasmonic acid, salicylic acid, abscisic acid, and ethylene. The genome-wide expression analysis suggests that about half of the rice MAPK genes are associated with pathogen infection and host defense response.

Ancient signals: comparative genomics of plant MAPK and MAPKK gene families.

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, and their components are encoded by highly conserved genes. The recent availability of genome sequences for rice and poplar now makes it possible to examine how well the previously described Arabidopsis MAPK and MAPKK gene family structures represent the broader evolutionary situation in plants, and analysis of gene expression data for MPK and MKK genes in all three species allows further refinement of those families, based on functionality. The Arabidopsis MAPK nomenclature appears sufficiently robust to allow it to be usefully extended to other well-characterized plant systems.

NOC/oFQ activates ERK and JNK but not p38 MAPK to impair prostaglandin cerebrovasodilation after brain injury.

Fluid percussion brain injury (FPI) elevates the CSF concentration of the opioid nociceptin/orphanin FQ (NOC/oFQ), which contributes to impairment of pial artery dilation to the prostaglandins (PG) PGE2 and PGI2. This study investigated the role of the ERK, p38, and JNK isoforms of mitogen-activated protein kinase (MAPK) in impaired PG cerebrovasodilation after FPI, and the relationship of brain injury induced release of NOC/oFQ to MAPK in such vascular impairment in newborn pigs equipped with a closed cranial window. FPI blunted PGE2 pial artery dilation, but U 0126 and SP 600125 (10(-6) M) (ERK and JNK MAPK inhibitors, respectively) partially prevented such impairment (7 +/- 1, 12 +/- 1, and 17 +/- 1 vs. 2 +/- 1, 3 +/- 1, and 5 +/- 1 vs. 4 +/- 1, 7 +/- 1, and 12 +/- 1% for 1, 10, and 100 ng/ml PGE2 in control, FPI, and FPI + U 0126 pretreated animals, respectively). In contrast, administration of SB 203580 (10(-5) M) (p38 MAPK inhibitor) did not prevent FPI impairment of PGE2 dilation. Co-administration of NOC/oFQ at the dose of 10(-10) M, the cerebrospinal fluid concentration observed after FPI, with PGE2 under non-brain injury conditions blunted PG dilation, but U 0126 or SP 600125 partially prevented such impairment (7 +/- 1, 11 +/- 1, and 16 +/- 2 vs. 0 +/- 1, 1 +/- 1, and 2 +/- 1, vs. 5 +/- 1, 9 +/- 1, and 13 +/- 2 for responses to PGE2 in control, NOC/oFQ, and NOC/oFQ + U 0126 treated animals, respectively). Administration of SB 203580 did not prevent impairment of PG pial artery dilation by NOC/oFQ. These data show that activation of ERK and JNK but not p38 MAPK contributes to impairment of PG cerebrovasodilation after FPI. These data suggest that NOC/oFQ induced ERK and JNK but not p38 MAPK activation contributes to impaired cerebrovasodilation to PG after FPI.

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes.

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.