Phosphorylation Of Multiple Proteins Research Articles

Capillary Isoelectric-Focusing Immunoassays to Study Dynamic Oncoprotein Phosphorylation and Drug Response to Targeted Therapies in Non–Small Cell Lung Cancer

Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non-small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEK inhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal-regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 μg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib and MEK1 inhibitor treatments in an NSCLC patient. NanoPro's higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPro's investigation for future drug development and evaluation of drug effects of targeted therapies.

Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

Abstract 5521: Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies.

Abstract Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC) and genomic analysis has been commonly used to identify patients who may benefit from targeted therapies. Nevertheless, the accuracy of predicting responses to targeted therapies is only 50-80% based on sequencing or FISH. Developing proteomic biomarkers is valuable for evaluating therapeutic effects and better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, low throughput or/and lack of assay reproducibility, accuracy and sensitivity. In this study, a novel capillary isoelectricfocusing (IEF) immunoassay system (ProteinSimple, Santa Clara, CA) was used to study the dynamic phosphorylation status of signaling molecules in tyrosine kinase or/and MEK inhibitor treated NSCLC cells. NanoPro showed the same dynamic Erk phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation enables multiple protein phosphorylation isoforms to be resolved and detected using pan-reactive antibodies. In PD325901 MEK inhibitor treated cells, a specific MEK response pattern to drug treatment was revealed by NanoPro. This pattern demonstrated on-target effect of the drug, which was not detectable by Western blotting. NanoPro also identified a MEK2 response signature that may be associated with NSCLC cell sensitivity to the tyrosine kinase inhibitor, erlotinib, and distinguished erlotinib-sensitive from erlotinib-resistant cells. This MEK2 signature was further confirmed in cells with acquired resistance to erlotinib when compared to respective parental cells. In an HCC827 xenograft study, Western blotting was not able to differentiate human cancer cell signals from mouse stromal cell interference. In contrast, NanoPro detected and distinguished human Erk1 isoforms from the mouse isoforms based on their pI differences and demonstrated that erlotinib effectively inhibited Erk phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. We further used NanoPro to monitor responses of protein phosphorylations to erlotinib and AZD6244 (a MEK1/2 inhibitor) in a NSCLC patient by profiling 18 signaling molecules with as little as 8 μg tumor material. Signals from baseline and inhibitor treated samples were precisely quantified and compared. In conclusion, NanoPro can assess protein phosphorylation both qualitatively and quantitatively to provide more information about the activation status of proteins, which is not readily assessed by traditional Western blotting. Its higher sensitivity, better reproducibility and reduced tissue requirement are appealing for drug development and evaluation of drug effects in targeted therapies. Citation Format: Jinqiu Chen, Jih-Hsiang Lee, Michelle Herrmann, Kang-Seo Park, Madeleine Heldman, Paul K. Goldsmith, Yisong Wang, Giuseppe Giaccone. Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5521. doi:10.1158/1538-7445.AM2013-5521

A nanofluidic immunoassay system to develop proteomic responsive biomarkers in non-small cell lung cancer.

27 Background: Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC). However, errors in predicting response to targeted therapies are 20-30%, based on sequencing or FISH, and patient specimen are at times not sufficient for conventional protein technologies. Developing clinically feasible proteomic biomarkers may be valuable to identify patients who may benefit from targeted therapy. Methods: NanoPro technology (ProteinSimple), is a capillary-based isoelectric-focusing (IEF) immunoassay system, which detects and quantifies multiple protein phosphorylation isoforms that are not readily assessed by traditional immunoassays. The platform only uses nanograms of protein for analysis. Results: We studied dynamic phosphoprotein activities in NSCLC cells (PC9, H827, H4006, H2122 and H322) treated with EGFR or/and MEK inhibitors. In MEK inhibitor (PD325901) treated cells, NanoPro showed that the drug efficiently inhibited Erk phosphorylation, and also revealed a complex MEK response profile which was not detectable by Western blotting, demonstrating its on-target effect. NanoPro also identified a MEK2 signature that associated with erlotinib sensitivity and distinguished erlotinib sensitive from resistant cells. This MEK2 signature was further confirmed in acquired resistant cells to erlotinib (H827R, H4006R). In a H827 xenograft study, NanoPro was able to detect and distinguish human Erk1 isoform from mouse Erk1 based on their pI difference, and clearly demonstrated that erlotinib effectively inhibited Erk phosphorylation in human xenograft cancer cells but not in surrounding mouse stromal cells. We further demonstrated that Nanopro could monitor erlotinib and AZD2644 response in a KrasG12C NSCLC patient (NCT01229150) by profiling 18 signaling molecules with as little as 4ug tumor material. Conclusions: NanoPro provides a valuable platform to monitor signaling response to targeted therapy. Nanopro assesses protein phosphorylation both qualitatively and quantitatively using small sample size, thus creates new opportunities for pharmacodynamic biomarker assessment in clinical studies.

Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor

Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

Sitagliptin: Anti-platelet effect in diabetes and healthy volunteers

Sitagliptin, a selective dipeptidyl peptidase-4 inhibitor drug is used to treat type-2 diabetes (T2DM). We investigated the anti-platelet activity of sitagliptin in patients with T2DM and in in vitro samples obtained from healthy humans. Patients with T2DM (27 male + 23 female) were selected and followed up before (control) and after treatment with sitagliptin for up to 3 months. Platelets were isolated from the blood of sitagliptin treated patients and controls. Patients with T2DM treated with sitagliptin for 1and 3 months, showed 10 ± 2% and 30 ± 5% inhibition of platelet aggregation, respectively. For the in vitro study, platelets from 10 normal humans (n = 10) were isolated. Platelet aggregation, intracellular free calcium and tyrosine phosphorylation of multiple proteins were measured by aggregometer, spectrofluorometer and western blotting, respectively. Platelets pre-treated with 5 and 10 µg/ml of sitagliptin, showed 25 ± 4% and 40 ± 6% inhibition of thrombin-induced platelet aggregation, respectively. Sitagliptin decreased intracellular free calcium (2.5-fold) and tyrosine phosphorylation of multiple proteins in thrombin-induced platelet activation. Sitagliptin inhibited platelet aggregation in T2DM as well as in healthy humans. Sitagliptin has significant concentration-dependent anti-platelet activity. This activity was due to its inhibitory effect on intracellular free calcium and tyrosine phosphorylation.

BCR/ABL modulates protein phosphorylation associated with the etoposide-induced DNA damage response

BCR/ABL expression is the key characteristic of chronic myeloid leukaemia (CML). The progression of CML is associated with genomic instability. Systematic analysis of DNA damage signalling in the presence of BCR/ABL thus offers opportunities to identify mechanisms of leukaemic progression. We therefore undertook a quantitative phosphoproteomics study to test whether BCR/ABL expression could globally affect the response to genotoxic stress signalling. Etoposide-induced DNA damage was chosen and the concentration and time of exposure determined such that apoptosis was not associated with the study. More than 1100 phosphoentities were identified. BCR/ABL was shown to significantly alter the response to etoposide in many cases. These include sites on MDC1, a key component of DNA repair, and Hemogen. Hemogen is a transcriptional target of HOXB4 and GATA1, two transcription factors involved in haemopoietic development, and is overexpressed in acute myeloid leukaemia. To validate Hemogen phosphorylation, absolute quantification using an isotopomeric standard and selected reaction monitoring was performed. This revealed a strong correlation with isobaric tagging data and offering quantification at about 10 fmol per million cells. Furthermore we found that multiple protein phosphorylation changes mediated by BCR/ABL were connected to the increased activation of NFκB, a key survival transcription factor, after etoposide exposure.

C-Abl tyrosine kinase in the DNA damage response: cell death and more

The robust cellular response to DNA damage (DNA damage response or DDR) comprises multiple intertwined signaling networks that function in concert to optimize the manner in which a cell reacts to genotoxic stress. Downstream DDR signaling depends on two main upstream activators—the PI3K-related protein kinases ATM and ATR, whose respective activation by distinct types of DNA damage (DD) leads to a rapid phosphorylation of multiple proteins involved in processes such as DNA repair, cell-cycle arrest and programmed cell death (reviewed in Jackson and Bartek1, Lavin2, Cimprich and Cortez3).

Abstract 4460: Mechanism of action of small molecule CXCR4 antagonists in human B-lymphoma cell lines

Abstract CXCR4 is a chemokine receptor frequently over-expressed on hematological cancers and solid tumors. CXCR4 and its ligand SDF1α (stromal derived factor-1α or CXCL12) play an important role in cancer metastasis, regulation of stem cell trafficking and neovascularization. We have observed previously that treatment with the small molecule CXCR4 antagonists Plerixafor (AMD3100, Mozobil) and AMD3465 in combination with Campath or Rituxan had a synergistic effect resulting in a prolongation of survival greater than that obtained with either agent alone in disseminated human B-lymphoma tumor models. We next explored the mechanism of action of CXCR4 antagonists in vitro using the human B-lymphoma cell lines Ramos, Raji, B104, BL31, IM9 or Wien133 which display similarly strong levels of cell surface CXCR4 expression. All cell lines responded to SDF1α but, in spite of comparable levels of CXCR4 expression, showed varying degrees of calcium influx and cell migration when stimulated. There was a good correlation between calcium influx and migratory responses for a given cell line. Addition of the CXCR4 antagonists Plerixafor or AMD3465 inhibited both responses. The compounds did not show any direct cytotoxicity and failed to inhibit spontaneous proliferation of Ramos, Raji and B104 human B-lymphoma cell lines. However, they did inhibit SDF1α-induced proliferation of these cell lines in agreement with their ability to block CXCR4. Moreover, CXCR4 antagonists inhibited SDF1α-induced phosphorylation of multiple proteins involved in cell migration, proliferation and survival signal pathways as well as some transcription factors, including Akt, ERK1/2, JNK, p70S6, p90RSK, GSK-3a/b, Tyk2, p38, MEK1, c-Jun, STAT3 and CREB. These results suggest that the CXCR4/SDF1α axis plays an important role in the migration, proliferation and survival of human B-lymphoma cells and that disruption of these functions with CXCR4 antagonists may contribute to their observed ability to enhance the anti-tumor activity of Campath and Rituxan in human B-lymphoma models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4460.

Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia–reperfusion injury and treatment

Protein phosphorylation plays a crucial role in the signal transduction pathways that regulate gene expression, metabolism, cell adhesion, and cell survival in response to oxidative stress. In this study, we have used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia–reperfusion injury. We show that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. A phosphoproteomics approach using anti-phosphotyrosine affinity purification and LC–MS/MS was then used to identify the protein targets of this stress-induced phosphorylation. Twenty-three tyrosine-phosphorylated proteins were identified, with the majority known to be associated with cell–cell junctions, the actin cytoskeleton, and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology, and survival. Importantly, Src kinase was shown to be a major upstream regulator of these events. Immunofluorescence studies, fluorescence-activated cell sorting, and cell-based assays were used to demonstrate oxidative stress-induced modification of cell adhesion structures and the cytoskeleton, induced de-adhesion, and increased apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1. These data demonstrate the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggest that targeting this kinase may be an effective strategy for preventing ischemia–reperfusion injury in the heart.

Role for ADAP in shear flow–induced platelet mechanotransduction

AbstractBinding of platelets to fibrinogen via integrin αIIbβ3 stimulates cytoskeletal reorganization and spreading. These responses depend on tyrosine phosphorylation of multiple proteins by Src family members and Syk. Among Src substrates in platelets is adhesion- and degranulation-promoting adapter protein (ADAP), an adapter with potential binding partners: SLP-76, VASP, and SKAP-HOM. During studies of platelet function under shear flow, we discovered that ADAP−/− mouse platelets, unlike ADAP+/+ platelets, formed unstable thrombi in response to carotid artery injury. Moreover, fibrinogen-adherent ADAP−/− platelets in shear flow ex vivo showed reduced spreading and smaller zones of contact with the matrix. These abnormalities were not observed under static conditions, and they could not be rescued by stimulating platelets with a PAR4 receptor agonist or by direct αIIbβ3 activation with MnCl2, consistent with a defect in outside-in αIIbβ3 signaling. ADAP+/+ platelets subjected to shear flow assembled F-actin–rich structures that colocalized with SLP-76 and the Rac1 exchange factor, phospho-Vav1. In contrast, platelets deficient in ADAP, but not those deficient in VASP or SKAP-HOM, failed to form these structures. These results establish that ADAP is an essential component of αIIbβ3-mediated platelet mechanotransduction that promotes F-actin assembly and enables platelet spreading and thrombus stabilization under fluid shear stress.

Leukocyte Ig-like Receptor B4 (LILRB4) Is a Potent Inhibitor of FcγRI-mediated Monocyte Activation via Dephosphorylation of Multiple Kinases

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of cell surface receptors that possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is believed to down-regulate activation signals mediated by non-receptor tyrosine kinase cascades through the recruitment of SHP-1. However, the exact mechanisms of LILRB4-mediated inhibition are not fully elucidated. In this study, we demonstrate high level surface expression of LILRB4 on THP-1 cells and primary peripheral blood monocytes, which profoundly inhibited production of a key pro-inflammatory cytokine (TNFalpha) induced by FcgammaRI (CD64). We also report that LILRB4 aggregated to sites of activation upon co-ligation with CD64 and that this may enhance its inhibitory effects. Cross-linking of CD64 on THP-1 cells markedly increased phosphorylation of multiple proteins including tyrosine kinases and signaling molecules (Lck, Syk, LAT, and Erk), an adaptor protein that targets protein-tyrosine kinases for degradation (c-Cbl) and a protein involved in the formation of actin cytoskeletal rearrangement (alpha-actinin-4). Co-ligation of LILRB4 considerably reduced CD64-mediated phosphorylation of Lck, Syk, LAT, Erk, and c-Cbl but not alpha-actinin-4, suggesting selective inhibition of signaling molecules. Treatment of cells with a broad-spectrum phosphatase inhibitor, sodium pervanadate (SP), significantly reversed LILRB4-mediated inhibition of TNFalpha production and protein tyrosine phosphorylation. In comparison, treatment with an SHP-1 specific inhibitor, sodium stibogluconate (SS) has no effects indicating involvement of phosphatase(s) other than SHP-1 in LILRB4 signaling. Collectively, our data show LILRB4 is a potent inhibitor of monocytes activation. This may provide a new potential therapeutic strategy for inflammatory conditions characterized by excessive TNFalpha production.

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.

Novel phosphotyrosine targets of FGFR2IIIb signaling

In partnership exclusively with the epithelial FGFR2IIIb isotype and a structurally-specific heparan sulfate motif, stromal-derived FGF7 delivers both growth-promoting and growth-limiting differentiation signals to epithelial cells that promote cellular homeostasis between stromal and epithelial compartments. Intercompartmental homeostasis supported by FGF7/FGFR2IIIb is subverted in many solid epithelial tumors. The normally mesenchymal-derived homologue FGFR1 drives proliferation and a progressive tumor-associated phenotype when it appears ectopically in epithelial cells. In order to understand the mechanism underlying the unique biological effects of FGFR2IIIb, we developed an inducible FGFR2IIIb expression system that is specifically dependent on FGF7 for activation in an initially unresponsive cell line to avoid selection for only the growth-promoting aspects of FGFR2IIIb signaling. We then determined FGF7/FGFR2IIIb signaling-specific tyrosine phosphorylated proteins within 5 min after FGF7 stimulation by phosphopeptide immunoaffinity purification and nano-LC-MS/MS. The FGF7/FGFR2 pair caused tyrosine phosphorylation of multiple proteins that have been implicated in the growth stimulating activities of FGFR1 that included multi-substrate organizers FRS2α and IRS4, ERK2 and phosphatases SHP2 and SHIP2. It uniquely phosphorylated CDK2 and phosphatase PTPN18 on sites involved in the attenuation of cell proliferation, and several factors that maintain nuclear-cytosolic relationships (emerin and LAP2), protein structure and other cellular fine structures as well as some proteins of unknown functions. Several of the FGF7/FGFR2IIIb-specific targets have been associated with maintenance of function and tumor suppression and disruption in tumors. In contrast, a number of pTyr substrates associated with FGF2/FGFR1 that are generally associated with intracellular Ca 2+-phospholipid signaling, membrane and cytoskeletal plasticity, cell adhesion, migration and the tumorigenic phenotype were not observed with FGF7/FGFR2IIIb. Our findings provide specific downstream targets for dissection of causal relationships underlying the distinct role of FGF7/FGFR2IIIb signaling in epithelial cell homeostasis.

Determination of phosphoproteins in higher plant thylakoids.

Redox-dependent thylakoid protein phosphorylation regulates both short- and long-term acclimation of the photosynthetic apparatus to changes in environmental conditions. The major thylakoid phosphoproteins belong to photosystem (PS)II (D1, D2, CP43, PsbH) and its light-harvesting antenna (Lhcb1, Lhcb2, CP29) but a number of minor phosphoproteins have also been identified. The detection methods traditionally include the radiolabeling techniques, electrophoretic separation of the phosphorylated and unphosphorylated forms of the protein, and the use of phosphoamino acid antibodies. The recent progress in mass spectrometry (MS) techniques and methods of proteomics allow for successful identification and analyses of protein phosphorylation. In mass spectrometry approaches, exogenous tracer is not needed and natural phosphorylation of proteins can be characterized with high sensitivity yielding the mapping of exact phosphorylation sites in the proteins as well. Various methods for detection of thylakoid phosphoproteins, including the preparation of phosphopeptides for mass spectrometric analyses and techniques for phosphopeptide identification by electrospray ionization MS are described. The experimental protocols for simultaneous identification of multiple phosphopeptides in complex peptide mixtures and for assess of stoichiometry for in vivo phosphorylation of multiple proteins are outlined.

Phospholipid Scramblase 1 Modulates a Selected Set of IgE Receptor-mediated Mast Cell Responses through LAT-dependent Pathway

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.

Microbial Products Activate Monocytic Cells through Detergent-Resistant Membrane Microdomains

Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.

Modulation of gold(III) porphyrin 1a-induced apoptosis by mitogen-activated protein kinase signaling pathways

Gold(III) porphyrin 1a is a novel gold(III) complex with selective anticancer effect in a number of human carcinoma cell lines. We have previously shown that gold(III) porphyrin 1a mediated mitochondrial transmembrane potential (ΔΨm) depletion, leading to cytochrome c release, nucleus translocation of apoptosis-inducing factor (AIF), and generation of reactive oxygen species (ROS). The current study addressed gold(III) porphyrin 1a-induced phosphoproteome alterations and modulation of cell death by the mitogen-activated protein kinase (MAPK) family proteins. ERK and p38MAPK were transiently activated upon gold(III) porphyrin 1a treatment. Inhibition of p38MAPK phosphorylation rescued gold(III) porphyrin 1a-induced cell death upstream of caspase activation. Attenuation of ΔΨm was the primary effect of gold(III) porphyrin 1a leading to p38MAPK phosphorylation. Further functional proteomic study suggested that differential regulation of phosphotyrosine proteins were related to p38MAPK activation in gold(III) porphyrin 1a-induced signal transduction cascade. In summary, p38MAPK modulated gold(III) porphyrin 1a-induced cell death downstream of mitochondria, and phosphorylation of multiple proteins also involved in this process. These results suggested that gold(III) porphyrin 1a is a promising anticancer agent directed toward the mitochondria.

XB130, a Novel Adaptor Protein for Signal Transduction

Adaptor proteins are important mediators in signal transduction. In the present study, we report the cloning and characterization of a novel adaptor protein, XB130. This gene is located on human chromosome 10q25.3 and encodes a protein of 818 amino acids. It contains several Src homology (SH)2- and SH3-binding motifs, two pleckstrin homology domains, a coiled-coil region, and a number of potential tyrosine or serine/threonine phosphorylation sites. Endogenous XB130 interacts with c-Src tyrosine kinase. Their co-expression in COS-7 cells resulted in activation of c-Src and elevated tyrosine phosphorylation of multiple proteins, including XB130 itself. XB130 expression in HEK293 cells enhanced serum response element- and AP-1-dependent transcriptional activation mediated by c-Src. XB130DeltaN, an N-terminal deletion mutant lacking a putative SH3-binding motif and several putative SH2-binding sites, reduced its ability to mediate Src signal transduction. Down-regulation of endogenous XB130 with siRNA reduced c-Src activity, IL-8 production, EGF-induced phosphorylation of Akt and GSK3beta, and altered cell cycles in human lung epithelial cells. These data suggest that XB130 as an adaptor may play an important role in the regulation of signal transduction and cellular functions.

CXCR3‐mediated T‐cell chemotaxis involves ZAP‐70 and is regulated by signalling through the T‐cell receptor

The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70,000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-gamma1 (PLCgamma1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCgamma1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCgamma1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function.