High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Analyses of genes encoding the Glycogen Synthase Kinases in rice and Arabidopsis reveal mechanisms which regulate their expression during development and responses to abiotic stresses.

Plant Glycogen Synthase Kinases (GSKs) enable a crosstalk among the brassinosteroid signaling and phytohormonal- and stress-response pathways to regulate various physiological processes. Initial information about regulation of the GSK proteins' activity was obtained, however, mechanisms that modulate expression of the GSK genes during plant development and stress responses remain largely unknown. Taking into account the importance of the GSK proteins, combined with the lack of in-depth knowledge about modulation of their expression, research in this area may provide a significant insight into mechanisms regulating these aspects of plant biology. In the current study, a detailed analysis of the GSK promoters in rice and Arabidopsis was performed, including identification of the CpG/CpNpG islands, tandem repeats, cis-acting regulatory elements, conserved motifs, and transcription factor-binding sites. Moreover, characterization of expression profiles of the GSK genes in different tissues, organs and under various abiotic stress conditions was performed. Additionally, protein-protein interactions between products of the GSK genes were predicted. Results of this study provided intriguing information about these aspects and insight into various regulatory mechanisms that influence non-redundant and diverse functions of the GSK genes during development and stress responses. Therefore, they may constitute a reference for future research in other plant species.

WRKY53 integrates classic brassinosteroid signaling and the mitogen-activated protein kinase pathway to regulate rice architecture and seed size.

In rice (Oryza sativa) and other plants, plant architecture and seed size are closely related to yield. Brassinosteroid (BR) signaling and the mitogen-activated protein kinase (MAPK) pathway (MAPK kinase kinase 10 [MAPKKK10]-MAPK kinase 4 [MAPKK4]-MAPK6) are two major regulatory pathways that control rice architecture and seed size. However, their possible relationship and crosstalk remain elusive. Here, we show that WRKY53 mediated the crosstalk between BR signaling and the MAPK pathway. Biochemical and genetic assays demonstrated that glycogen synthase kinase-2 (GSK2) phosphorylates WRKY53 and lowers its stability, indicating that WRKY53 is a substrate of GSK2 in BR signaling. WRKY53 interacted with BRASSINAZOLE-RESISTANT 1(BZR1); they function synergistically to regulate BR-related developmental processes. We also provide genetic evidence showing that WRKY53 functions in a common pathway with the MAPKKK10-MAPKK4-MAPK6 cascade in leaf angle and seed size control, suggesting that WRKY53 is a direct substrate of this pathway. Moreover, GSK2 phosphorylated MAPKK4 to suppress MAPK6 activity, suggesting that GSK2-mediated BR signaling might also regulated MAPK pathway. Together, our results revealed a critical role for WRKY53 and uncovered sophisticated levels of interplay between BR signaling and the MAPK pathway in regulating rice architecture and seed size.

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi.

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.

Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Phosphorylation of the Gα protein Gpa2 promotes protein kinase A signaling in yeast.

Heterotrimeric G proteins are important molecular switches that facilitate transmission of a variety of signals from the outside to the inside of cells. G proteins are highly conserved, enabling study of their regulatory mechanisms in model organisms such as the budding yeast Saccharomyces cerevisiae Gpa2 is a yeast Gα protein that functions in the nutrient signaling pathway. Using Phos-tag, a highly specific phosphate binding tag for separating phosphorylated proteins, we found that Gpa2 undergoes phosphorylation and that its level of phosphorylation is markedly increased upon nitrogen starvation. We also observed that phosphorylation of Gpa2 depends on glycogen synthase kinase (GSK). Disrupting GSK activity diminishes Gpa2 phosphorylation levels in vivo, and the purified GSK isoforms Mck1 and Ygk3 are capable of phosphorylating Gpa2 in vitro Functionally, phosphorylation enhanced plasma membrane localization of Gpa2 and promoted nitrogen starvation-induced activation of protein kinase A. Together, the findings of our study reveal a mechanism by which GSK- and nutrient-dependent phosphorylation regulates subcellular localization of Gpa2 and its ability to activate downstream signaling.

Brassinosteroid Regulates Root Development with Highly Redundant Genes in Hexaploid Wheat.

Brassinosteroid (BR) plays an important role in plant development and biotic and abiotic stress tolerance, but its specific function remains largely unknown in wheat (Triticum aestivum L.), preventing its utilization in this important crop. In this study, the function of BR and its underlying cytological role in wheat root development were comprehensively investigated. Our findings demonstrated that BR has a conserved function in regulating root length in wheat, and novel roles in regulating lateral root emergence and root diameter were uncovered. Analyses of BR homologous gene composition and evolutionary divergence demonstrated that the genetic framework of the wheat BR pathway was close to that of rice, but contained highly redundant homologous copies of genes from the subgenome A, B and D. These homologous copies showed active expression and shared a conserved BR response. The expression of wheat DWF4 and glycogen synthase kinase (GSK) genes in Arabidopsis confirmed that multiple homologous copies maintained their conserved function in regulating root development, highlighting their redundant status and indicating that a special challenge exists in wheat gene modification to deal with this high redundancy. However, our results suggested that the hypermorphic effect of T. aestivum GSK (TaGSK) genes with point mutations may be an effective approach to overcome this redundancy in the manipulation of BR signaling in wheat. Our study provides fundamental data uncovering the function of BR in wheat root development, the underlying genetic basis and a possible strategy to manipulate BR signaling in hexaploid wheat.

Histone acetyltransferase MoHat1 acetylates autophagy-related proteins MoAtg3 and MoAtg9 to orchestrate functional appressorium formation and pathogenicity in Magnaporthe oryzae.

Macroautophagy/autophagy is critical for normal appressorium formation and pathogenicity of the rice blast fungus Magnaporthe oryzae, but the molecular base of autophagy linked to pathogenicity remains elusive in this or other pathogenic fungi. We found that MoHat1, a histone acetyltransferase (HAT) homolog, had a role in the regulation of autophagy through the acetylation of autophagy related proteins MoAtg3 and MoAtg9. We also found that MoHat1 was subject to regulation by the protein kinase MoGsk1 that modulated the translocation of MoHat1 from the nucleus to the cytoplasm with the assistance of MoSsb1, a protein chaperone. The alternation of intracellular location affected MoHat1 in the modification of cytosolic autophagy proteins that maintained normal autophagy. Furthermore, we provided evidence linking acetylation of MoAtg3 and MoAtg9 by MoHat1 to functional appressorium development and pathogenicity. Together with the first report of MoAtg9 being subject to acetylation regulation by MoHat1, our studies depicted how MoHat1 regulated autophagy in conjunction with MoGsk1 and how normal autophagy was linked to appressorium formation and function and pathogenicity of M. oryzae. Abbreviations: A/Ala: alanine; AP: autophagosome; Atg genes/proteins: autophagy-related genes/proteins; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; DAPI: 4', 6-diamidino-2-phenylindole; D/Asp: aspartic acid; GFP: green fluorescent protein; GSK3: glycogen synthase kinase 3; HAT: histone acetyltransferase; Hsp70: heat-shock protein 70; IH: invasive hyphae; K/Lys: lysine; MMS: methyl methanesulfonate; Mo: Magnaporthe oryzae; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; R/Arg: arginine; S/Ser: serine; T/Thr: threonine; TOR: target of rapamycin; WT: wild type; YFP: yellow fluorescent protein.

Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase is an archetypal multifunctional moonlighting protein involved in diverse cellular processes including metabolism, insulin signaling, proliferation, differentiation, apoptosis, neuronal function and embryonic development. The two known isoforms, GSK-3α and GSK-3ß that undergo activation/inactivation by post-translational, site-specific phosphorylation incorporate a vast number of substrates in their repertoire. Dysregulation of GSK-3 has been linked to diverse disease entities including cancer. The role of GSK-3 in cancer is paradoxical and enigmatic. The enzyme functions as a tumour promoter or suppressor based on the context, cell type and phosphorylation status. GSK-3 is the central hub that orchestrates signals from the Wnt/ß-catenin, PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, hedgehog, Notch and TP53 pathways to elicit regulatory influences on cancer initiation, epithelial-mesenchymal transition, and resistance to therapy. As a direct target of several microRNAs, GSK-3 influences hallmark attributes of cancer, cancer stemness and treatment resistance. There is overwhelming evidence to indicate that GSK-3 is aberrantly regulated in different cancer types. Consequently, GSK-3 has emerged as a potential therapeutic target in cancer. A plethora of natural and synthetic GSK-3 modulators have been discovered and the number of patents published for GSK-3 inhibitors has also been steadily increasing in recent years. This review focuses on the intricate interactions between GSK-3 and oncogenic signalling circuits as well as the feasibility of targeting GSK-3 for the treatment of cancer.

Eugenosedin-A improves glucose metabolism and inhibits MAPKs expression in streptozotocin/nicotinamide-induced diabetic rats.

This study examined the effects of eugenosedin-A (Eu-A) in a streptozotocin (STZ)/nicotinamide-induced rat model of type II diabetes mellitus (T2DM). Six-week-old Sprague-Dawley rats were randomly divided into three groups: (1) RD group, normal rats fed a regular diet (RD), (2) DM group, T2DM rats fed a high-fat diet, and (3) Eu-A group, T2DM rats fed a high fat diet plus oral Eu-A (5 mg/kg/day). After 30 days, the DM group had higher body weight, higher blood glucose and lower insulin levels than the RD group. The DM group also had increased protein expression of glycogen synthase kinase (GSK) in liver and skeletal muscle and decreased protein expression of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), IRS-2, AMP-activated protein kinase (AMPK), glucose transporter-4 (GLUT-4), glucokinase (GCK), and peroxisome proliferator-activated receptor γ (PPAR-γ). STZ/nicotinamide-induced T2DM increased the expression of mitogen-activated protein kinases (MAPKs: p38, ERK, JNK) and inflammatory p65 protein. In the Eu-A treated T2DM rats, however, blood glucose was attenuated and the insulin concentration stimulated. Changes in IR, IRS-1 and IRS-2 proteins as well as AMPK, GLUT-4, GCK, GSK, PPAR-γ, MAPKs, and inflammatory p65 proteins were ameliorated. These results suggested that Eu-A alleviates STZ/nicotinamide-induced hyperglycemia by improving insulin levels and glucose metabolism, and inhibiting the MAPKs- and p65-mediated inflammatory pathway.

MicroRNA-203 suppresses proliferation in liver cancer associated with PIK3CA, p38 MAPK, c-Jun, and GSK3 signaling.

Primary liver cancer (hepatocellular carcinoma, HCC) is a leading cause of cancer-related deaths, and alternative ways to treat this disease are urgently needed. In recent years, novel approaches to cancer treatment have been based on microRNAs, small non-coding RNA molecules that play a crucial role in cancer progression by regulating gene expression. Overexpression of some microRNAs has shown therapeutic potential, but whether or not this was the case for microRNA-203 (miR-203) in liver cancer was unknown. Therefore, the aim of this study was to investigate the effect of miR-203 overexpression in liver cancer and explore the related mechanisms. Liver cancer cells from the HepG2 and Hep3B cell lines were transfected with either miR-203 mimics or negative control RNA, and then the cells were subjected to cell viability, cell proliferation, and Western blotting assays. As a result of microRNA-203 overexpression, HepG2 and Hep3B cell viability and cell proliferation significantly declined. Furthermore, microRNA-203 overexpression led to inhibited expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3)/protein kinase B (Akt), c-Jun, and p38 mitogen-activated protein kinases (p38 MAPK), and restored glycogen synthase kinase 3 (GSK 3) activity in HepG2 cells. Our results suggest that c-Jun, p38 MAPK, PIK3CA/Akt, and GSK3 signaling involved in the effect of miR-203 on the proliferation of HCC cells.

Alcohol Regulates BK Surface Expression via Wnt/ß-Catenin Signaling.

It has been suggested that drug tolerance represents a form of learning and memory, but this has not been experimentally established at the molecular level. We show that a component of alcohol molecular tolerance (channel internalization) from rat hippocampal neurons requires protein synthesis, in common with other forms of learning and memory. We identify ß-catenin as a primary necessary protein. Alcohol increases ß-catenin, and blocking accumulation of ß-catenin blocks alcohol-induced internalization in these neurons. In transfected HEK293 cells, suppression of Wnt/ß-catenin signaling blocks ethanol-induced internalization. Conversely, activation of Wnt/ß-catenin reduces BK current density. A point mutation in a putative glycogen synthase kinase phosophorylation site within the S10 region of BK blocks internalization, suggesting that Wnt/ß-catenin directly regulates alcohol-induced BK internalization via glycogen synthase kinase phosphorylation. These findings establish de novo protein synthesis and Wnt/ß-catenin signaling as critical in mediating a persistent form of BK molecular alcohol tolerance establishing a commonality with other forms of long-term plasticity. SIGNIFICANCE STATEMENT: Alcohol tolerance is a key step toward escalating alcohol consumption and subsequent dependence. Our research aims to make significant contributions toward novel, therapeutic approaches to prevent and treat alcohol misuse by understanding the molecular mechanisms of alcohol tolerance. In our current study, we identify the role of a key regulatory pathway in alcohol-induced persistent molecular changes within the hippocampus. The canonical Wnt/ß-catenin pathway regulates BK channel surface expression in a protein synthesis-dependent manner reminiscent of other forms of long-term hippocampal neuronal adaptations. This unique insight opens the possibility of using clinically tested drugs, targeting the Wnt/ß-catenin pathway, for the novel use of preventing and treating alcohol dependency.

CREBH Couples Circadian Clock With Hepatic Lipid Metabolism.

The circadian clock orchestrates diverse physiological processes critical for health and disease. CREB, hepatocyte specific (CREBH) is a liver-enriched, endoplasmic reticulum (ER)-tethered transcription factor known to regulate the hepatic acute phase response and energy homeostasis under stress conditions. We demonstrate that CREBH is regulated by the circadian clock and functions as a circadian regulator of hepatic lipid metabolism. Proteolytic activation of CREBH in the liver exhibits typical circadian rhythmicity controlled by the core clock oscillator BMAL1 and AKT/glycogen synthase kinase 3ß (GSK3ß) signaling pathway. GSK3ß-mediated phosphorylation of CREBH modulates the association between CREBH and the coat protein complex II transport vesicle and thus controls the ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian manner. Functionally, CREBH regulates circadian expression of the key genes involved in triglyceride (TG) and fatty acid (FA) metabolism and is required to maintain circadian amplitudes of blood TG and FA in mice. During the circadian cycle, CREBH rhythmically regulates and interacts with the hepatic nuclear receptors peroxisome proliferator-activated receptor α and liver X receptor α as well as with the circadian oscillation activator DBP and the repressor E4BP4 to modulate CREBH transcriptional activities. In conclusion, these studies reveal that CREBH functions as a circadian-regulated liver transcriptional regulator that integrates energy metabolism with circadian rhythm.

Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

Glycogen synthase kinase is a regulator of the circadian clock of Neurospora crassa.

Timekeeping by circadian clocks relies upon precise adjustment of expression levels of clock proteins. Here we identify glycogen synthase kinase (GSK) as a novel and critical component of the circadian clock of Neurospora crassa that regulates the abundance of its core transcription factor white collar complex (WCC) on a post-transcriptional level. We show that GSK specifically binds and phosphorylates both subunits of the WCC. Reduced expression of GSK promotes an increased accumulation of WC-1, the limiting factor of the WCC, causing an acceleration of the circadian clock and a shorter free-running period.

Molecular pathways for glucose homeostasis, insulin signaling and autophagy in hepatitis C virus induced insulin resistance in a cellular model.

Chronic HCV infection induces insulin resistance (IR). We studied this in a persistently infected cell line with defects in glucose homeostasis resulting from the phosphorylation of glycogen synthase (GS Ser641) and GS kinase isoform 3ß (GSK 3ßSer9). Reversal of these effects in cells cured of HCV with interferon supports viral specificity. Insulin signaling was disrupted by IRS-1 Ser312 phosphorylation and dysregulation of the Akt pathway. In infected cells, active autophagy was revealed by the formation of LC3 puncta or by increased levels (50-200%) of the markers Beclin 1 and conjugated Atg5/Atg12. Inhibition of autophagy by 3-methyl-adenine (3-MA) reduced Beclin1 levels, inhibited IRS-1 Ser312 or GS Ser641 phosphorylation and decreased viral load. Furthermore, IRS-1 Ser312 and Beclin1 were co-immunoprecipitated suggesting that they interact. It thus appears that HCV infection disturbs glucose homeostasis or insulin signaling to induce IR and also elicits autophagy that may contribute to this process.

An active part of Artemisia sacrorum Ledeb. suppresses gluconeogenesis through AMPK mediated GSK3ß and CREB phosphorylation in human HepG2 cells.

In this study, we investigated the effects of a petroleum ether fraction of Artemisia sacrorum Ledeb. (Compositae) (PEASL) on glucose production through AMP-activated protein kinase (AMPK) activation in human HepG2 cells. PEASL significantly inhibited glucose production in a concentration-dependent manner, and this effect was reversed in the presence of compound C, a selective AMPK inhibitor. PEASL markedly induced the phosphorylation of AMPK and downstream acetyl-CoA carboxylase (ACC) in a time- and concentration-dependent manner. In addition, it markedly increased the phosphorylations of glycogen synthase kinase 3ß (GSK3ß) in a concentration-dependent manner. In contrast, cAMP response element binding protein (CREB), a key transcription factor for gluconeogenic enzyme phosphorylation, decreased in a concentration-dependent manner. PEASL downregulated the gluconeogenesis gene expression of peroxisome proliferation activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in a concentration-dependent manner. In addition, the gene expression of orphan nuclear receptor small heterodimer partner (SHP) increased, also in a concentration-dependent manner. These effects were also abolished by pretreatment with compound C, an AMPK inhibitor. This indicates that PEASL inhibited glucose production via the AMPK-GSK-CREB pathway in HepG2 cells, and these effects appeared to be capable of revealing anti-diabetic mechanism of PEASL in HepG2 cells.

Invasion of HeLa cells by group B streptococcus requires the phosphoinositide-3-kinase signalling pathway and modulates phosphorylation of host-cell Akt and glycogen synthase kinase-3.

The group B streptococcus (GBS) is an opportunistic bacterial pathogen with the ability to cause invasive disease. While the ability of GBS to invade a number of host-cell types has been clearly demonstrated, the invasion process is not well understood at the molecular level. What has been well established is that modulation of host-cell actin microfilaments is essential for GBS invasion to occur. Phosphoinositide-3 kinase (PI3K) is a key regulator of the cytoskeleton in eukaryotic cells. Our goal in this investigation was to explore the role of the PI3K/Akt signalling pathway in epithelial cell invasion by GBS. The epithelial cell invasion process was mimicked using the HeLa 229 cell-culture model. Treating HeLa cells with chemical inhibitors of PI3K, Akt or Ras prior to bacterial infection inhibited GBS invasion but not attachment; treatment with 30 microM LY294002 (PI3K inhibitor) reduced GBS invasion by 75%, 20 microM L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (ICIO) (Akt inhibitor) reduced GBS invasion by 50%, and 10 microM manumycin A (Ras inhibitor) inhibited GBS invasion by 90%. Genetic inactivation of the p85alpha or p110alpha PI3K subunits in HeLa cells also reduced GBS invasion by 55 and 30%, respectively. Western blot analysis revealed that phosphorylation of host-cell Akt and glycogen synthase kinase-3 (GSK-3) occurs in response to GBS infection, and that this is mediated upstream by PI3K. Infection of HeLa cells with GBS triggers pro-survival signalling and protects the HeLa cells from camptothecin-induced caspase-3 cleavage. The results from this investigation show that GBS both requires and activates the PI3K/Akt host-cell signalling pathway during invasion of epithelial cells.

T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses.

T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (Oryza sativa glycogen synthase kinase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsis brassinosteroid insensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes.

Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag.

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.