Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Identification and characteristic analysis of an extracellular signal-regulated kinase from Ostrinia furnacalis Guenée.

The extracellular signal-regulated kinase (ERK) pathway, a critical genetic determinant, controls diverse physiological functions, including innate immunity, development, and stress response. In the current study, a full-length cDNA (1592bp) encoding the ERK gene (OfERK) was cloned from Ostrinia furnacalis Guenée (GenBank accession number: MF797866). The open reading frame of the OfERK gene encoded 364 amino acids and shared 96.43%-98.08% amino acid identities with other insect mitogen-activated protein kinases. For spatiotemporal analysis of the expression pattern, OfERK exhibited a significant peak expression on the 3rd day of the pupa stage and showed the highest expression in hemocytes specifically. Indirect immunofluorescence assays and immuno-electron microscopy revealed a wide distribution of the OfERK protein in hemocytes and epidermis. Moreover, the results demonstrated that the Bt Cry1Ab-activated toxin significantly induces the expression of OfERK. Other genes related to immune response, development, and stress response exhibited dynamic changes in expression after Cry1Ab oral treatment. The expression of OfERK was downregulated through RNA interference, and the correlation of its expression with other related genes was verified using quantitative real-time polymerase chain reaction. Our study provides valuable insights into the regulatory mechanism of ERK in insects for future studies.

The selective regulation of immune responses by matrix metalloproteinase MMP14 in Ostrinia furnacalis.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune-related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection-responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune-related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double-stranded RNA and bacterial infection.

JAK/STAT signaling pathway and its regulation on insect immunity.

JAK/STAT signaling pathway is involved in important biological processes, such as cell growth, differentiation, apoptosis and immune regulation, and is one of the most important signal transduction pathways mediated by cytokines. JAK/STAT is a relatively conserved signaling pathway in insects, which is the main immune pathway together with Toll signaling pathway and Imd signaling pathway to resist the invasion of pathogens. It plays a vital role in insect immunity, hormone regulation and other physiological regulation processes. In this review, we summarize the cytokine receptor superfamily, JAKs family, STATs family, JAK/STAT signaling pathway and its mechanism of negative feedback regulation. We analyze the important functions and the latest research progress of JAK/STAT signaling pathway in insect infected by parasites, viruses and fungi. Finally, we propose the remaining problems in the study of JAK/STAT signaling pathway, so as to provide direction and reference for further research in this field.

Characterization and functional analysis of a myeloid differentiation factor 88 in Ostrinia furnacalis Guenée larvae infected by Bacillus thuringiensis.

Myeloid differentiation factor 88 (MyD88) is a pivotal adapter protein involved in activating nuclear factor NF-κB of the Toll pathway in insect innate immunity. MyD88 has been extensively studied in vertebrates and Drosophila. However, the information ascribed to MyD88 in Lepidoptera is scarce. In the present study, an Ostrinia furnacalis MyD88 (OfMyD88) cDNA was cloned and functionally characterized (GenBank accession no. MN906311). The complete cDNA sequence of OfMyD88 is 804 bp, and contains a 630 bp open reading frame encoding 209 amino acid residues. OfMyD88 has the death domain (DD), an intermediate domain, and the Toll/interleukin 1 receptor (TIR) domain. OfMyD88 was widely expressed in immune-related tissues such as hemocytes, fat body, midgut, and integument, with the highest expression level in hemocytes, and the lowest expression level in integument. To clarify the immune function of MyD88, O. furnacalis larvae were challenged with Bacillus thuringiensis (Bt) through feeding. Bt oral infection had significantly up-regulated the expression of OfMyD88 and immune genes, including PPO2 (prophenoloxidase 2), Attacin, Gloverin, Cecropin, Moricin, GRP3 (ß-1, 3-Glucan recognition protein 3), and Lysozyme, and increased the activities of PO and lysozyme in hemolymph of O. furnacalis larvae. Knockdown of OfMyD88 by RNA interference suppressed the expression levels of immune related genes, but not PPO2 in the larvae orally infected with Bt, suggesting that OfMyD88 is involved in defending against Bt invasion through the Toll signaling pathway, but does not affect the PPO expression in O. furnacalis larvae.

Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway.

Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.

Transcriptomic analysis provides insights into the immune responses and nutrition in Ostrinia furnacalis larvae parasitized by Macrocentrus cingulum.

Macrocentrus cingulum is a principal endoparasite of Ostrinia furnacalis larvae. M. cingulum larvae repress host immune responses for survival and ingest host nutrients for development until emerging. However, most investigations focused on the mechanisms of how wasps repress the host immunity, the triggered immune responses and nutrient status altered by wasps in host are neglected. In this study, we found that parasitized O. furnacalis larvae activated fast recognition responses and produced some effectors such as lysozyme and antimicrobial peptides, along with more consumption of trehalose, glucose, and even lipid to defend against the invading M. cingulum. However, the expression of peroxidase 6 and superoxide dismutase 2 (SOD 2) was upregulated, and the messenger RNA (mRNA) levels of cellular immunity-related genes such as thioester-containing protein 2 (TEP 2) and hemocytin were also reduced, suggesting that some immune responses were selectively shut down by wasp parasitization. Taken together, all the results indicated that parasitized O. furnacalis larvae selectively activate the immune recognition response, and upregulate effector genes, but suppress ROS reaction and cellular immunity, and invest more energy to fuel certain immune responses to defend against the wasp invading. This study provides useful information for further identifying key components of the nutrition and innate immune repertoire which may shape host-parasitoid coevolutionary dynamics.

Characterization and functional analysis of a Relish gene from the Asian corn borer, Ostrinia furnacalis (Guenée).

Pathogen-induced host immune responses reduce the efficacy of pathogens used to control pests. However, compared to the well-deciphered immunity system of Drosophila melanogaster, the immunity system of agricultural pests is largely unconfirmed through functional analysis. Beginning to unveil mechanisms of transcription regulation of immune genes in the Asian corn borer, Ostrinia furnacalis, we cloned the complementary DNA (cDNA) of a transcription factor Relish by rapid amplification of cDNA ends. The 3164 bp cDNA, designated Of-Relish, encodes a 956-residue protein. Bioinformatic analysis showed that Of-Relish had a Rel homology domain, a predicted cleavage site between Q409 and L410 , six ankyrin repeats, and a death domain. The response of Of-Relish expression to the Gram-negative bacteria Pseudomonas aeruginosa was sooner and stronger than to the Gram-positive Micrococcus luteus. The antimicrobial peptide genes Attacin and Gloverin had similar expression patterns in response to the infections. Knockdown of Of-Relish led to a decrease in Attacin and Gloverin messenger RNA levels, suggesting that Attacin and Gloverin were regulated by Of-Relish. Together, the results suggested that Of-Relish is a key component of the IMD pathway in O. furnacalis, involved in defense against P. aeruginosa through activation of Attacin and Gloverin.

FKBP39 controls nutrient dependent Nprl3 expression and TORC1 activity in Drosophila.

Target of Rapamycin Complex 1 (TORC1) is a master regulator that coordinates nutrient status with cell metabolism. The GTPase-activating protein towards Rags complex 1 (GATOR1) inhibits TORC1 activity and protects cells from damage during periods of stress. Here we characterize multiple pathways that regulate the expression of the GATOR1 component Nprl3 in Drosophila. We determine that the stability of Nprl3 is impacted by the Unassembled Soluble Complex Proteins Degradation (USPD) pathway. In addition, we find that FK506 binding protein 39 (FKBP39)-dependent proteolytic destruction maintains Nprl3 at low levels in nutrient replete conditions. Nutrient starvation abrogates the degradation of the Nprl3 protein and rapidly promotes Nprl3 accumulation. Consistent with a role in promoting the stability of a TORC1 inhibitor, mutations in fkbp39 decrease TORC1 activity and increase autophagy. Finally, we show that the 5'UTR of nprl3 transcripts contain a functional upstream open reading frame (uORF) that inhibits main ORF translation. In summary, our work has uncovered novel mechanisms of Nprl3 regulation and identifies an important role for FKBP39 in the control of cellular metabolism.

Royal jelly attenuates metabolic defects in a Drosophila mutant with elevated TORC1 activity.

Target of rapamycin complex 1 (TORC1) is a master regulator of cell metabolism, and its dysregulation has been linked to an array of pathologies, including cancer and age-related diseases. Nprl3, a component of GTPase-activating protein towards Rags complex 1 (GATOR1), inhibits TORC1 activity under nutrient scarcity status. The nprl3 mutant exhibits some metabolic defects due to hyper TORC1 activity in Drosophila Royal jelly (RJ) is a honeybee-secreted product and plays an essential role in caste differentiation that requires TORC1 activity. RJ is also used as a health-benefit food for its potential roles on antioxidant and anti-aging. In this study, nprl3-mutant flies were used to measure the effect of RJ on metabolic modulation. Interestingly, RJ feeding significantly increased survival and decreased TORC1 activity in the nprl3 mutant. RJ feeding also ameliorated the abnormal reactive oxygen species (ROS) levels and energy status in the nprl3 mutant. The proteins in RJ were characterized to be the essential components in increasing nprl3 mutant viability. These findings suggest that RJ modulates some metabolic defects associated with elevated TORC1 activity and that the nprl3-mutant fly might be a useful tool for investigating the bioactive components of RJ in vivo.

Transcription Analysis of the Stress and Immune Response Genes to Temperature Stress in Ostrinia furnacalis.

Ostrinia furnacalis is one of the most important pests on maize. O. furnacalis larvae are frequently exposed to the temperature challenges such as high temperature in summer and cold temperature in winter in the natural environment. High and low temperature stress, like any abiotic stress, impairs the physiology and development of insects. Up to now, there is limited information about gene regulation and signaling pathways related to the high and cold stress response in O. furnacalis. High-throughput sequencing of transcriptome provides a new approach for detecting stress and immune response genes under high and low temperature stresses in O. furnacalis. In the present study, O. furnacalis larvae were treated with the temperature at 8 and 40°C, and the responses of O. furnacalis larvae to the temperature stress were investigated through RNA-sequencing and further confirmation. The results showed that immune responses were up-regulated in larvae by the cold stress at 8°C while some stress response genes, such as HSP family, GST-2, Bax inhibitor and P450, were significantly increased at 40°C. Furthermore, quantitative real time polymerase chain reaction were performed to quantify the expression levels of immune related genes, such as PGRP-LB, antimicrobial peptides, lysozyme, serine protease and stress response genes such as small HSPs and HSP90, and the expression levels of these genes were similar to the RNA-seq results. In addition, the iron storage protein Ferritin was found to be involved in the response to temperature stress, and the changes of total iron concentration in the hemolymph were, in general, consistent with the expression levels of Ferritin. Taken together, our results suggested that the stress response genes were involved in the defense against the heat stress at 40°C, and the immune responses triggered by cold stress might provide protection for larvae from cold stress at 8°C. More interestingly, our results showed that during the responses to temperature stress, the total iron concentration in hemolymph regulated by Ferritin increased, which might help O. furnacalis in surviving the low and high temperature stress.

Clip domain prophenoloxidase activating protease is required for Ostrinia furnacalis Guenée to defend against bacterial infection.

The prophenoloxidase (PPO) activating system in insects plays an important role in defense against microbial invasion. In this paper, we identified a PPO activating protease (designated OfPAP) containing a 1203 bp open reading frame encoding a 400-residue protein composed of two clip domains and a C-terminal serine protease domain from Ostrinia furnacalis. SignalP analysis revealed a putative signal peptide of 18 residues. The mature OfPAP was predicted to be 382 residues long with a calculated Mr of 44.8 kDa and pI of 6.66. Multiple sequence alignment and phylogenetic analysis indicated that OfPAP was orthologous to the PAPs in the other lepidopterans. A large increase of the transcript levels was observed in hemocytes at 4 h post injection (hpi) of killed Bacillus subtilis, whereas its level in integument increased continuously from 4 to 12 hpi in the challenged larvae and began to decline at 24 hpi. After OfPAP expression had been silenced, the median lethal time (LT50) of Escherichia coli-infected larvae (1.0 day) became significantly lower than that of E. coli-infected wild-type (3.0 days, p < 0.01). A 3.5-fold increase in E. coli colony forming units occurred in larval hemolymph of the OfPAP knockdown larvae, as compared with that of the control larvae not injected with dsRNA. There were notable decreases in PO and IEARase activities in hemolymph of the OfPAP knockdown larvae. In summary, we have demonstrated that OfPAP is a component of the PPO activation system, likely by functioning as a PPO activating protease in O. furnacalis larvae.

ß-1,3-Glucan recognition protein 3 activates the prophenoloxidase system in response to bacterial infection in Ostrinia furnacalis Guenée.

Pattern recognition receptors (PRRs) are biosensor proteins that bind to non-self pathogen associated molecular patterns (PAMPs). ß-1,3-glucan recognition proteins (ßGRPs) play an essential role in immune recognition and signaling pathway of insect innate immunity. Here, we report the cloning and characterization of cDNA of OfßGRP3 from Ostrinia furnacalis larvae. The OfßGRP3 contains 1455 bp open reading frame, encoding a predicted 484 amino acid residue protein. In hemocytes, the expression levels of OfßGRP3 in Escherichia coli-challenged group were higher than those of Bacillus subtilis-challenged group at 2, 4, 8, 10 and 12 h post injection (HPI). In fat body, OfßGRP3 expression in both B. subtilis and E. coli-challenged group was significantly higher than that in untreated group from 4 to 10 HPI, and then the expression continuously dropped from 12 to 36 HPI. The OfßGRP3 expression in laminarin-injected group was higher than that in lipopolysaccharides (LPS)-injected group in various test tissues from 4 to 24 HPI. The LT50 of E. coli-infected OfßGRP3-RNAi larvae (1.0 days) was significantly lower compared with that of E. coli infected wild-type larvae (3.0 days) (p < 0.01). Only 10.2% Sephadex G50 beads (degree 3) were completely melanized in the larvae inoculated with OfßGRP3 dsRNA, as compared to 48.8% in control larvae (p < 0.01). A notable reduction in the PO activity and IEARase activity in hemolymph was also detected in the OfßGRP3 knockdown larvae. Our study demonstrates that OfßGRP3 is one of PRR members involved the PPO-activating system in O. furnacalis larvae.

Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system.

Serine protease inhibitors of the serpin superfamily are regulators of proteases involved in a variety of physiological processes including immune responses. In this study, we have isolated a full-length serpin cDNA from Ostrinia furnacalis. The 1188 bp open reading frame encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. Ofserpin1 contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E(344) to S(353)) and a predicted P1-P1' cleavage site (Leu(360)-Ser(361)). Ofserpin1 mRNA and protein were detected in all the tested tissues, particularly in hemocytes and integument. The recombinant protein inhibited chymotrypsin and trypsin in a dose-dependent manner, and were significantly cleaved by the enzyme trypsin and chymotrypsin. Ofserpin1 impeded the prophenoloxidase activation cascade by 45.6% at 16.5 µg, and affected activity of prophenoloxidase activating protease. Levels of Ofserpin1 transcripts in the integument were higher than those in hemocytes, fat body and midgut. After an immune challenge with Staphylococcus aureus and Escherichia coli, the relative mRNA levels of Ofserpin1 decreased in 2-10h post-infection (hpi) in integument and hemocytes compared to the untreated control. Our results suggested that Ofserpin1 has serine protease inhibitory activity and is likely involved in the regulation of prophenoloxidase activation system in O. furnacalis.

Parasitization by Macrocentrus cingulum (Hymenoptera: Braconidae) influences expression of prophenoloxidase in Asian corn borer Ostrinia furnacalis.

A prophenoloxidase (PPO) cDNA (OfPPO) was cloned from the Asian corn borer Ostrinia furnacalis. Sequence analysis revealed a full length transcript of the OfPPO cDNA with 2,686 bp, containing a 2,079 bp open reading frame (ORF), a 73-bp 5'-untranslated region, and a 534-bp 3'-untranslated region with a poly(A) signal. The ORF encodes a 693-amino acid polypeptide, containing two distinct copper-binding regions, a plausible thiol ester site, two proteolytic activation sites, and a conserved C-terminal region, but lacks a signal peptide sequence. Expression of the OfPPO transcript in the plasma, hemocytes, fat body and midgut was inhibited by Macrocentrus cingulum at 4 h post-parasitization (pp). In situ hybridization analysis showed that the hemocytes, especially the oenocytoids, hybridized strongly with the DNA probes of the OfPPO gene. No signal was detected in the cuticular epithelium or fat body of the parasitized larvae. Colloidal gold particles were used to visualize the PPO by immunoelectron microscopy. The time course study revealed a decrease in the labeling of the OfPPO at 4, 6, 8, 12, and 1 day pp in the larval integument and midgut parasitized by M. cingulum. We infer from time course studies of OfPPO gene expression and PO enzymatic activity that OfPPO in the integument is released from hemocytes and that the OfPPO expression was influenced at the transcriptional, translational, and then the post-translational level by parasitization challenge.

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae.

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA). Both Mg(2+) and Cu(2+) stimulated PO activity when compared with controls. The beta-sheet content of PPO treated with Mg(2+) and Cu(2+) increased significantly (P<0.05). The purified PPO has magnesium level of 5.674+/-2.294 microg/mg and copper level of 1.257+/-0.921 microg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.

Identification of genes differentially regulated by transcription factor, AP-2delta.

In an effort to better understand the transcriptional program regulated by transcription factor, AP-2delta, we used cDNA microarray to evaluate the relative expression of human genes in AD293 cells by exogenous expression of AP-2delta. Microarray showed 29 genes that were up-regulated and 39 genes with a down-regulated expression pattern. Among the identified genes were those encoding transcription factors, signal transduction molecules, kinases, as well as genes regulating cell growth, differentiation, and cell adhesion, a set of genes suggested to be regulated by AP-2. The results of microarray was validated for some regulated genes by real-time PCR analysis. These observations shed novel insight into the mechanism of AP-2delta action and provide a range of target genes for further investigation.

Dual specificity phosphotase 18, interacting with SAPK, dephosphorylates SAPK and inhibits SAPK/JNK signal pathway in vivo.

The SAPK/JNKs play important roles in numerous cellular processes, and for this reason they have become putative drug targets. Most dual-specificity protein phosphatases (DSPs) play important roles in the regulation of mitogenic signal transduction and cell cycle control in response to extracellular stimuli. Dual-specificity phosphatase 18 (DUSP18), a newly recognized SAPK/JNK phosphatase, is widely expressed. This expression is modulated in response to extracellular stimuli. By phosphorylation assay, pull down and coimmunoprecipitation experiments, it is shown here that DUSP18 interacts with SAPK/JNK and dephosphorylates it both in vitro and in vivo. DUSP18 does not dephosphorylate p38 or p44ERK1. Furthermore, DUSP18 inhibits SAPK/JNK pathway in vivo. Based on these findings, DUSP18 appears to serve an important role by regulation of SAPK/JNK pathway.

Characterization of a novel human protein phosphatase 2C family member, PP2Ckappa.

A novel member of human protein phosphatase 2C gene named PP2Ckappa was isolated from a human fetal brain cDNA library. The 2.0 kb cDNA encodes a 372 amino acid polypeptide with an intact protein phosphatase 2C (PP2C) catalytic domain. Reverse transcription-PCR (RT-PCR) revealed that the PP2Ckappa was widely expressed in normal human tissues. Transient transfection suggested that PP2Ckappa was localized in the nucleus in AD293 cells. Recombinant Trx-His-PP2Ckappa showed phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing phospho-threonine residues. Furthermore, the overexpression of PP2Ckappa distinctly activated the heat shock transcription factor pathway in eukaryotic cells.

Cloning and characterization of a novel splice variant of human U2AF1L3 gene.

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3' splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.