Protein Phosphorylation In Signal Transduction Research Articles

Novel peanut-specific human IgE monoclonal antibodies enable screens for inhibitors of the effector phase in food allergy.

Background10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms.ObjectiveWe used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells.MethodsTwo human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8.ResultsPeanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation.ConclusionAllergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.

Exploring the Molecular Mechanism of Zhi Bai Di Huang Wan in the Treatment of Systemic Lupus Erythematosus Based on Network Pharmacology and Molecular Docking Techniques

Objective: To investigate the molecular mechanism and simulated validation of Zhi Bai Di Huang Pill (ZBDHP) for the treatment of systemic lupus erythematosus (SLE) using network pharmacology and molecular docking techniques. Methods: The active ingredients of ZBDHP were obtained through the TCMSP database and the Canonical SMILES of the active ingredients were queried through Pubchem. The targets of the active ingredients were predicted in the SwissTarget database based on the SMILES. The SLE-related disease targets were obtained through the GeneCards, OMIM and DisGenets databases, and the intersection targets of ZBDHP and SLE were obtained using the Venny 2.1.0 online platform. Intersection targets build a visual protein interaction network (PPI) through the STRING database, and the core targets were identified by network topology analysis. GO analysis and KEGG pathway enrichment analysis of the intersecting targets were performed using the DAVID database. Finally, the molecular docking of the first four active ingredients and the first four core target genes were verified by Pubchem, the PDB database and CB-Dock online molecular docking technology. Results: ZBDHP screened 91 potential active ingredients and 816 potential targets. Among them, 141 genes were intersected by ZBDHP and SLE. The network topology analysis showed that the main active ingredients were Hydroxygenkwanin, Alisol B, asperglaucide, Cerevisterol, etc., and the key target genes were TNF, AKT1, EGFR, STAT3, etc. GO and KEGG enrichment analysis showed that common targets interfere with biological processes or molecular functions such as signal transduction protein phosphorylation, inflammatory response, transmembrane receptor protein tyrosine kinase activity, etc., through multiple signaling pathways, such as pathways in cancer, Kaposi sarcoma-associated herpesvirus infection, the PI3K-Akt signaling pathway, lipid and atherosclerosis, hepatitis B, etc. Molecular docking results showed that the active components of ZBDHP have good binding activity to the core targets of SLE. Conclusions: This study reveals that the ZBDHP treatment of SLE is a complex mechanistic process with multi-components, multi-targets and multi-pathways, and it may play a therapeutic role in SLE by inhibiting the production, proliferation and apoptosis of inflammatory factors. In conclusion, the present study provides a theoretical basis for further research on ZBDHP.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Lipoprotein(a) Induces Human Aortic Valve Interstitial Cell Calcification

Lipoprotein(a), or Lp(a), significantly increased alkaline phosphatase activity, release of phosphate, calcium deposition, hydroxyapatite, cell apoptosis, matrix vesicle formation, and phosphorylation of signaltransduction proteins; increased expression of chondro-osteogenic mediators; and decreased SOX9and matrix Gla protein (p<0.001). Inhibition of MAPK38 and GSK3β significantly reduced Lp(a)-induced calcification of human aortic valve interstitial cells (p< 0.001). There was abundant presence of Lp(a) and E06 immunoreactivity in diseased human aortic valves. The present study demonstrates a causal effect forLp(a) in aortic valve calcification and suggests that interfering with the Lp(a)pathway couldprovide anovel therapeutic approach in the management of this debilitating disease.

Co-culture Activation of MAP Kinase in Drosophila S2 Cells.

Intercellular communication often involves phosphorylation of signal transduction proteins, including mitogen-activated protein kinases (MAPKs). Immunological detection of phosphorylated MAPK can be used to monitor signaling in vivo, identify novel pathway components, and assess ligand activity. In this chapter, I describe a cell co-culture method to assess activity of cell-bound extracellular ligands that result in phosphorylation of the ERK (extracellular signal-regulated kinase) MAPK in Drosophila. This protocol may be adaptable to other pathways and/or model systems.

Gel-based comparative phosphoproteomic analysis on rice embryo during germination.

Seed germination is a well regulated process, which incorporates many events including signal transduction, mobilization of reserves, reactive oxygen species scavenging and cell division. Although many transcriptomic and proteomic studies have been conducted on this process, regulation of protein modification has not been studied. To better understand the mechanism, a gel-based comparative phosphoproteomic study was performed on rice embryo during the germination process. In total, 168 protein spots exhibited significantly changed Pro-Q staining intensity during germination. Using matrix-assisted laser deionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis, 193 proteins were identified. By combining Pro-Q and Coomassie brilliant blue stain intensity analyses, 109 proteins were verified to be phosphorylation regulation proteins. Functional analyses indicated that phosphorylation of proteins involved in stress response and storage was gradually enhanced. Phosphorylation of signal transduction proteins was mainly activated during the early stage of germination, while stress response and storage protein phosphorylation were enhanced at the late stage. Enzyme assays proved that the phosphorylation of fructokinase, pyruvate kinase, malate dehydrogenase, GDP-mannose 3,5-epimerase1, ascorbate peroxidase and glutathione S-transferase could consistently enhance their activity. This study showed the dynamic changes of protein phosphorylation status in rice embryo during germination and provided new insight into understanding the mechanism underlying this process.

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

BackgroundAmphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation.ResultsCathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.ConclusionsSingle-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

Novel 6-bp Deletion Mutation in visfatin Gene and Its Associations with Birth Weight and Bodyweight in Chinese Cattle

Visfatin, like insulin, induces phosphorylation of signal transduction proteins that operatate downstream of the insulin receptor. The present study is focused on detecting deletion of visfatin gene and analyzing its effect on growth traits in six Chinese cattle breeds (Nangyang, Luxi, Qinchuan, Jiaxian Red, Grassland Red, and Chinese Holstein) using DNA sequencing, PCR-SSCP and PCR-RFLP methods. For the first time, a 6-bp deletion of visfatin was described and two alleles were revealed: W and D. The χ2-test analysis demonstrated that all breeds were in agreement with Hardy-Weinberg equilibrium (Pgt;0.05). The associations of the novel 6-bp deletion of visfatin gene with growth traits of Nanyang cattle at 6-, 12-, 18-, and 24-mon-old were analyzed. Birth weight, 12- and 24-mon-old cattle with genotype WW had greater birth weight and average daily gain than genotype WD (P<0.01 or P<0.05). These results suggest that the deletion may influence the birth weight and bodyweight in 12 mon-old cattle.

Cell-Penetrant, Nanomolar O-GlcNAcase Inhibitors Selective against Lysosomal Hexosaminidases

SummaryPosttranslational modification of metazoan nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) is essential, dynamic, and inducible and can compete with protein phosphorylation in signal transduction. Inhibitors of O-GlcNAcase, the enzyme removing O-GlcNAc, are useful tools for studying the role of O-GlcNAc in a range of cellular processes. We report the discovery of nanomolar OGA inhibitors that are up to 900,000-fold selective over the related lysosomal hexosaminidases. When applied at nanomolar concentrations on live cells, these cell-penetrant molecules shift the O-GlcNAc equilibrium toward hyper-O-GlcNAcylation with EC50 values down to 3 nM and are thus invaluable tools for the study of O-GlcNAc cell biology.

Phosphorylation and lipid raft association of fibroblast growth factor receptor‐2 in oligodendrocytes

Fibroblast growth factors (FGFs) and their receptors (FGFRs) initiate diverse cellular responses that contribute to the regulation of oligodendrocyte (OL) function. To understand the mechanisms by which FGFRs elicit these cellular responses, we investigated the phosphorylation of signal transduction proteins and the role of cholesterol-glycosphingolipid-enriched "lipid raft" microdomains in differentiated OLs. Surprisingly, we found that the most abundant tyrosine-phosphorylated protein in OLs was the 120-kd isoform of FGFR2 and that it was phosphorylated even in the absence of FGF2, suggesting a potential ligand-independent function for this receptor. Furthermore, FGFR2, but not FGFR1, was associated with lipid raft microdomains in OLs and myelin (but not in astrocytes). This provides the first evidence for the association of FGFR with TX-100-insoluble lipid raft fractions. FGFR2 phosphorylated the key downstream target, FRS2 in OLs. Raft disruption resulted in loss of phosphorylated FRS2 from lipid rafts, coupled with the loss of Akt but not of Mek or Erk phosphorylation. This suggests that FGFR2-FRS2 signaling in lipid rafts operates via the PI3-Kinase/Akt pathway rather than the Ras/Mek/Erk pathway, emphasizing the importance of microenvironments within the cell membrane. Also present in lipid rafts in OLs and myelin, but not in astrocytes, was a novel 52-kd isoform of FGFR2 that lacked the extracellular ligand-binding region. These results demonstrate that FGFR2 in OLs and myelin possess unique characteristics that are specific both to receptor type and to OLs and provide a novel mechanism to elicit distinct cellular responses that mediate both FGF-dependent and -independent functions.

Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

Epidermal Growth Factor and/or Growth Hormone Induce Differential, Side‐Specific Signal Transduction Protein Phosphorylation in Enterocytes

Epidermal growth factor (EGF) plus growth hormone (GH) enhances luminal glutamine transport into rabbit and human intestinal cells. Our objective was to screen for activation status of signal proteins in C2(BBe)1 cells (enterocyte-like cell line) in response to side-specific EGF or GH treatment and to investigate the dependence of EGF receptor (EGFR) phosphorylation status on its tyrosine kinase. C2(BBe)1 cells on Transwells were treated for 15 minutes on either the basolateral or apical-side with EGF or GH. Lysates underwent Kinetworks phospho site-screen-2.1 analysis (duplicate experiments). In addition, lysates from cells treated as above with or without tyrphostin AG1478 (a specific EGFR tyrosine kinase inhibitor) underwent Western blot analysis for total EGFR and EGFR phosphorylated on tyrosine 1173, 1086 or 1068 (4-7 experiments). Kinetworks phospho-screening demonstrated a broad range of interactions dependent on both side of exposure and protein studied. From this screen, it appears that ErbB2, Met, and insulin receptor (R)/insulin-like growth factor 1 R are not involved in the growth factors signals. For EGFR phosphorylation, basolateral, but not apical, EGF was a strong activator. Synergism was seen, but only with apical EGF plus basolateral GH. All EGFR phosphorylations were EGFR tyrosine kinase dependent. In contradistinction, apical EGF phosphorylated FAK and MAPKs. Kinetworks phosphoprotein screens can suggest pathways involved in side-specific and synergistic interaction between EGF and GH. For EGFR, synergism by EGF + GH was noticed only with Ap EGF plus Bl GH and was EGFR tyrosine kinase dependent. Adaptive intestinal responses due to enterally administrated EGF might be accelerated by the availability of parenteral GH.

P-329 Activated c-Met in lung cancer leads to dramatic changes in cell motility/migration, reactive oxygen species formation, phosphorylation of signal transduction proteins

P-329 Activated c-Met in lung cancer leads to dramatic changes in cell motility/migration, reactive oxygen species formation, phosphorylation of signal transduction proteins

Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Effect of Recombinant Interleukin-6 and Thrombopoietin on Isolated Guinea Pig Bone Marrow Megakaryocyte Protein Phosphorylation and Proplatelet Formation

ABSTRACT: Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

Isolation and characterisation of Kasumi-1 human myeloid leukaemia cell line resistant to tumour necrosis factor alpha-induced apoptosis.

Tumour necrosis factor (TNF)-alpha induces apoptosis in a human acute myeloid leukaemia cell line, Kasumi-1. To examine the role of protein phosphorylation in signal transduction of TNF-alpha-induced apoptosis, a variant cell line resistant to TNF-alpha was established by an intermittent challenge of Kasumi-1 cells with increasing concentrations of TNF-alpha for 6 months. The mechanism of resistance to TNF-alpha appears to be in the post-receptor pathway because expression of p55 TNF receptor in the variant cells is increased compared with that of the parental Kasumi-1 cells. In renaturation assays, TNF-alpha induced a rapid activation of different protein kinases of different molecular weights, including the 50 kDa protein kinase (PK50) followed by the 35 kDa protein kinase (PK35), in the parental Kasumi-1 cells. The dose-response of TNF-alpha required to activate PK50 and PK35 was closely related to concentrations of TNF-alpha that induced apoptosis. Treatment of Kasumi-1 cells with ceramide also activated PK35. In TNF-resistant variant cells, activation of PK35 in response to TNF-alpha or ceramide was practically nil. These findings suggest that activation of PK35 through the ceramide pathway may play an important role in signal transduction of TNF-alpha in the Kasumi-1 cell line, while the decreased activation of PK35 may explain the insensitivity of the variant cells towards TNF-alpha.

Photoreceptor light-adaptation mediated by S-modulin, a member of a possible regulatory protein family of protein phosphorylation in signal transduction

In vertebrate retinal photoreceptors, cytoplasmic [Ca 2+] decreases upon exposure to light. A Ca 2+-binding protein, S-modulin, detects this [Ca 2+] decrease and reduces the light-sensitivity of the cell to induce light-adaptation. The reduction of the sensitivity is attained by disinhibition or facilitation of rhodopsin phosphorylation, a quenching mechanism of light-activated rhodopsin. S-modulin-like proteins are found in the brain as well. Several of these proteins show similar S-modulin effects, suggesting that these proteins also participate in the regulation of protein phosphorylation in the signal transduction in their host cells.

Calcium-dependent induction of novel proteins by abscisic acid in wheat aleurone tissue of different developmental stages

Aleurone tissue of mature wheat (Triticum aestivum L. cv. Sappo) grains make novel polypeptides in response to abscisic acid (ABA), but only in the presence of Ca(2+). Effects of ABA plus Ca(2+) include up- and down-modulation of other polypeptides. The ABA-induced polypeptides appear not to be the 21-kilodalton (kDa) amylase inhibitor which has been reported to be ABA-inducible in barley.Aleurone tissue from developing grains of different ages failed to respond to ABA plus Ca(2+) in any way. Endogenous ABA levels were determined by monoclonal radioimmunoassay in developing, mature, and "sensitised" developing tissues. The ABA level rose to a maximum at 35 days post anthesis but was not detectable in mature cells. Developing layers sensitised to gibberellic acid (GA) showed decreased levels of ABA, similar to those in mature tissue, concurrent with acquired responsiveness to GA in respect of its induction of α-amylase. However, these sensitised cells still remained non-responsive to added ABA in terms of modulation of polypeptide pattern, though they did respond to ABA in the blocking of GA-induced α-amylase production. The role of protein phosphorylation in signal transduction was examined. The implications of these findings are discussed with reference to the role of ABA in developing and mature aleurone cells.