Phosphorylation Of Isoforms Research Articles

Phosphorylation of CDK2 on threonine 160 influences silencing of sex chromosome during male meiosis.

In mammalian meiosis, the X and Y chromosomes are largely unsynapsed and transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation), forming a specialized nuclear territory called sex or XY body. An increasing number of proteins and noncoding RNAs were found to localize to the sex body and take part in influencing expression of sex chromosome genes. Cyclin-dependent kinase 2 (Cdk2 (-/-)) spermatocytes show incomplete sex chromosome pairing. Here, we further showed that phosphorylation of CDK2 isoform 1 (p-CDK2(39) [39 kDa]) on threonine 160 localizes to the sites of asynapsis and the sex body, interacting with phosphorylated gamma-H2AX. Meanwhile, p-CDK2(39) is frequently mislocalized throughout the sex body, and meiotic sex chromosome inactivation is disrupted in PWK×C57BL/6J hybrid mice. Furthermore, pachytene spermatocytes treated with mevastatin (an inhibitor of p-CDK2) showed overexpression of sex chromosome-linked genes. Our results highlight an important role for p-CDK2(39) in influencing silencing of the sex chromosomes during male meiosis by interacting with gamma-H2AX.

The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters

Precise homoeostasis of the intracellular concentration of Cl− is achieved via the co-ordinated activities of the Cl− influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.

Phosphorylation of Arabidopsis transketolase at Ser428 provides a potential paradigm for the metabolic control of chloroplast carbon metabolism

Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin–Benson–Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser(41) of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca(2+) release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser(41). This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca(2+) homeostasis.

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment.

Detection of protein phosphorylation and charge isoforms using vertical one-dimensional isoelectric focusing gels.

During many biological responses, changes in protein modifications (e.g., phosphorylation) are often more critical than changes in protein abundance in determining the outcome of cellular responses. These important regulatory changes can alter a protein's location, activity, or binding partners. Monitoring modifications such as phosphorylation is often impeded, or even prevented, because of the need for specialized reagents and equipment that are expensive and/or time-consuming to produce. However, many protein modifications alter the isoelectric point (pI) of a protein. Therefore, we developed a denaturing, one-dimensional isoelectric focusing (IEF) procedure that separates proteins based on their pI to resolve different isoforms, allowing a relatively simple strategy for detecting changes in protein modifications. Although similar results can be achieved by two-dimensional gel electrophoresis, the method described here uses a multi-well SDS-PAGE format that allows many more samples to be assayed within a single gel, thereby greatly decreasing both the time and cost needed to assess modifications of a single protein in response many different treatment conditions. To increase the sensitivity of detection, we also optimized a procedure to transfer proteins from these gels to membranes for subsequent immunodetection. This combination of techniques provides the means of interrogating the number and stoichiometry of isoforms from total protein extracts without a priori knowledge of which modification may occur.

Abstract A40: Detection of Erk1/2, Mek1/2, and Akt isoform phosphorylation in tumors of 115 patients with different cancer types using the NanoPro™1000 technology.

Abstract Proteomic analysis of cellular signaling networks has strongly gained in importance in the field of cancer research and treatment. In particular, there is a focus on the activation status of growth factor receptor related signaling cascades represented by phosphoproteins as key mediators of cellular signaling. For example, the inhibition of the EGF-receptor pathway has been widely integrated into clinical practice for different tumor entities, e.g. non small lung cancer and colorectal cancer. Although, predictive genetic markers of response to targeted therapy such as KRAS testing are already in use, heterogeneity in responses to such treatment has been clinically observed. In order to understand this heterogeneity on the proteome level, the activation status of cell signaling proteins has to be analyzed. In this study, we differentially analyzed basal isoform phosphorylation of selected key proteins from two EGF-receptor downstream pathways in breast cancer (n=26), non-small cell lung cancer (n=29), colorectal cancer (n= 30) and liver metastases of colorectal cancer (n= 30) using the NanoPro1000™ technology. Patient's samples were collected according to Indivumed's standard operating procedures, ensuring a high quality of tissues characterized by ischemia times below 10 minutes, comprehensive clinical data and corresponding sets of body fluids. The detailed characterization of isoform phosphorylation was conducted using established NanoPro1000™ protocols for Erk1/2, Mek1/2 and Akt. Statistical analysis of data revealed that isoform phosphorylation of Erk1/2, Mek1/2 and Akt significantly differed among individual patients, but not between tumor entities. Differences in phosphorylation patterns were characterized by the degree of overall phosphorylation, as well as individual patterns of isoform phosphorylation. This indicates that the in-depth analysis of isoform phosphorylation can reveal significant biological differences and might result in therapy relevant, predictive signatures independent of the mutation status. Hence, we established cut-off values for basal signaling molecule phosphorylation, to potentially enable the selection of patients, who will most likely benefit from targeted treatment. Based on the cut-off values we classified patients into statistical significant groups of high, moderate and low signaling molecule phosphorylation. In next steps, this classification will be further validated in terms of clinical relevance to response to targeted therapy. In summary, the NanoPro1000™ technology can be used to robustly screen a large cohort of patients for signaling molecule phosphorylation. The analysis of patients on the proteome level may be helpful to identify predictive biomarker for the development of resistance to anti-EGF-receptor treatment and thus further improve personalized medicine. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A40. Citation Format: Florian T. Unger, Jana Krüger, Janina Schaller, Giese Rebecca, Cordula Dede, Alexandra Samsen, Hartmut Juhl, Kerstin A. David. Detection of Erk1/2, Mek1/2, and Akt isoform phosphorylation in tumors of 115 patients with different cancer types using the NanoPro™1000 technology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A40.

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.

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

BackgroundScaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.Methodology/Principal FindingsWe have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.Conclusions/SignificanceOur results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

Hypoxia alters phosphorylation status of insulin‐like growth factor (IGF)‐binding protein‐1 and attenuates biological activities of IGF‐I in HepG2 cell cultures

Insulin-like growth factor (IGF)-I is known to stimulate fetal growth. One of the IGF-binding proteins, IGFBP-1, suppresses IGF-I activity, and thereby inhibits fetal growth. Because hypoxic stress in the uterus is known to cause fetal growth restriction, we examined the effects of hypoxia on IGFBP-1 production and phosphorylation status. Because liver is a main IGFBP-1 production site in the fetus, we used a hepatoma cell line, HepG2 cells, that secrete a large amount of IGFBP-1, express IGF-I receptors and model fetal liver metabolism in vitro. IGFBP-1 was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (PAGE) following immunoblotting, and IGFBP-1 phosphorylation status was analyzed by native PAGE following immunoblotting. Total concentrations of IGFBP-1 in media were higher and the highly phosphorylated isoforms were dominant in low oxygen conditions. Phosphorylation of IGF-I receptor by IGF-I was attenuated in low oxygen conditions. IGF-I-induced phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated in low oxygen conditions as well. However, attenuated phosphorylation of IGF-I receptor and IRS-1 were not observed in low oxygen conditions if the cells were stimulated with LR³IGF-I that has a similar binding affinity to IGF-I receptor but much less binding affinity to IGFBP-1 compared to those of native IGF-I. While IGF-I-induced cell proliferation was also inhibited in low oxygen conditions, LR³IGF-I-stimulated cell proliferation was not inhibited. These findings indicate that low oxygen conditions inhibit IGF-I action by increasing IGFBP-1, especially phosphorylated IGFBP-1, which inhibits IGF-I action. This study has indicated that hypoxia-induced IGFBP-1 production in the fetus may be a conserved physiological mechanism for restricting IGF-I-stimulated fetal growth.

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction-associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N-benzyl-p-toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze-clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non-stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction-induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways.

Abstract 2408: Comparison of ERK, AKT and MEK isoform phosphorylations between primary and corresponding metastatic colorectal carcinoma lesions using the NanoPro1000 technology.

Abstract Proteomic analysis of cellular signaling networks has strongly gained in importance in the field of cancer research and treatment. In particular, there is a focus on the activation status of growth factor receptor related signaling cascades represented by phosphoproteins, as key mediators of cellular signaling. For example, the inhibition of the EGF-receptor pathway has been widely integrated into clinical practice for different tumor entities, e.g. non small lung cancer and colorectal cancer. Although, predictive genetic markers of response to targeted therapy such as KRAS testing are already in use, heterogeneity in responses to treatment has been clinically observed. In order to understand this heterogeneity on the proteome level, the activation status of cell signaling proteins has to be analyzed. The in-depth analysis of isoform phosphorylation can potentially reveal significant biological differences in activation patterns and might result in predictive signatures. In this study, we differentially analyzed basal isoform phosphorylations of selected key proteins of two EGF-receptor downstream pathways, in four primary colorectal tumors and their corresponding metastases using the NanoPro1000 technology. Patient's biospecimen were collected according to standard operating procedures, ensuring a high quality of tissues characterized by ischemia times below 10 minutes, comprehensive clinical data and corresponding sets of body fluids. The detailed characterization of isoform phosphorylations was conducted using the recently introduced NanoPro1000 technology. This assay provides the separation of phospho protein isoforms by high-resolution isoelectric focusing (IEF) combined with a specific, antibody based detection. In addition to these data, the mutation status of selected, clinical relevant genes (KRAS, BRAF and PIK3CA) was determined in primary and metastatic tumor samples using Sanger sequencing. Among the analyzed primary tumors, there were two KRAS mutants, one BRAF mutant and one wild type tumor. Identical results were found in the matched metastasis. NanoPro1000 analysis revealed that isoform phosphorylation of ERK1/2, AKT and MEK1/2 differed among patients, as well as primary tumors and metastases. For example, ERK1 showed a tendency to be higher mono- and dual-phosphorylated in the primary tumors, whereas mono-phosphorylated ERK2 was elevated in metastases. However, a correlation to the mutation status was not found. These differences may explain the heterogeneity of individual responses to target anticancer treatment among patients, independent of their mutation status. Therefore, the NanoPro1000 technology can potentially be useful to identify predictive biomarker for the development of resistance to anti-EGF-receptor treatment and further improve personalized medicine. Citation Format: Florian T. Unger, Jana Krueger, Janina Schaller, Cordula Dede, Alexandra Samsen, Hartmut Juhl, Kerstin A. David. Comparison of ERK, AKT and MEK isoform phosphorylations between primary and corresponding metastatic colorectal carcinoma lesions using the NanoPro1000 technology. [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 2408. doi:10.1158/1538-7445.AM2013-2408

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

St. John's wort relieves pain in an animal model of migraine

Despite the substantial improvement that antimigraine drugs brought to migraineurs, there is the need for a long-acting and better tolerated migraine treatment than actual pharmacotherapy. St. John's wort (SJW), a medicinal plant endowed with a favourable tolerability profile, showed numerous bioactivities. We here investigated the pain-relieving property of SJW and its main components, hypericin and flavonoids, in a mouse model induced by nitric oxide (NO) donors administration. The NO donors nitroglycerin and sodium nitroprusside (SNP) induced allodynia (cold plate test) and hyperalgesia (hot plate test). Western blotting experiments were performed to detect c-Fos and protein kinase C (PKC) expression within periaqueductal grey matter (PAG). A single oral administration of an SJW dried extract (5 mg/kg p.o.) produced a prolonged relief from pain hypersensitivity. Similarly, preventive SJW administration increased the latency to the induction of hyperalgesia and reduced the duration of the painful symptomatology. Among SJW main components, hypericin showed a similar profile of activity, whereas flavonoids were devoid of any antihyperalgesic effect. To clarify the cellular pathways involved in the SJW mechanism of action, we examined the effects induced by the herbal drug on PKC. NO donors' administration upregulated and increased phosphorylation of PKCγ and PKCε isoforms within PAG that was prevented by SJW treatment. The absence of behavioural side effects or altered animals' locomotor activity by SJW was demonstrated. These results suggest SJW as a safe therapeutic perspective for migraine pain, and indicate PKC as an innovative target for antimigraine therapy.

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.

The Role of Extracellular Signal-Related Kinase During Abdominal Aortic Aneurysm Formation

It is hypothesized that activation of extracellular signal-related kinase (ERK) is critical in activating matrix metalloproteinases (MMPs) during abdominal aortic aneurysm (AAA) formation. C57BL/6 male mice underwent either elastase or heat-inactivated elastase aortic perfusion (n = 9 per group). Mouse aortic smooth muscle cells were transfected with ERK-1 and 2 siRNA along with or without elastase treatment. Mouse and human aortic tissue were analyzed by Western blots, zymograms, and immunohistochemistry, and statistical analysis was done using Graphpad and Image J softwares. Western blot and immunohistochemistry documented increased phospho-mitogen-activated protein kinase kinase-1/2 (pMEK-1/2; 153%, p = 0.270 by Western) and pERK (171%, p = 0.004 by Western blot) in the elastase perfused aortas. Male ERK-1(-/-) mice underwent elastase perfusion, and aortic diameter was determined at day 14. ERK-1(-/-) mice failed to develop AAA, and histologic analysis depicted intact collagen and elastin fibers in the aortas. Zymography of aortas of elastase-treated ERK-1(-/-) mice showed lower levels of proMMP2 (p < 0.005) and active MMP2 (p < 0.0001), as well as proMMP9 (p = 0.037) compared with C57BL/6 mice. siRNA transfection of ERK-1 and -2 significantly reduced formation of pro- and active MMP2 (p < 0.01 for both isoforms) in aortic smooth muscle cells treated with elastase in vitro. Human AAA tissue had significantly elevated levels of pMEK-1/2 (150%, p = 0.014) and pERK (159%, p = 0.013) compared with control tissues. The MAPK (mitogen-activated protein kinase)/ERK pathway is an important modulator of MMPs during AAA formation. Targeting the ERK pathway by reagents that inhibit either the expression or phosphorylation of ERK isoforms could be a potential therapy to prevent AAA formation.

Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

BackgroundThe present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles.ResultsIn denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2–10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2–16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3–13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2–18 fold in atrophic denervated muscle.ConclusionsThe results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

P2.19 Aaptamine, Demethyloxyaaptamine, and Isoaaptamine: A Proteomic-Based Screening of Protein Targets in Cisplatin-Resistant Tumor Cells

ABSTRACT Marine sponges are a rich source of natural compounds that possess potent anticancer activities. In continuation of our research of aaptamines - bioactive marine sponge metabolites - we investigated the alkaloid aaptamine (1) and two natural derivatives, demethyloxyaaptamine (2), and isoaaptamine (3), previously isolated from the marine sponge Aaptos sp., and shown to have promising anticancer properties via a largely undefined mode of action ( Fig. 1 ). All three alkaloids showed equal levels of cytotoxicity in vitro against a cisplatin-sensitive human embryonal carcinoma cell line, NTERA2, and NTERA2-R, a cisplatin-resistant subline derived from this cell line. We investigated the effect of the alkaloids on NTERA2-R cells at IC50 (concentration causing 50 % reduction of trypan blue excluding cells after 48 h, compared to untreated control). Aaptamine caused significant inhibition of cell proliferation and induction of a G2/M cell cycle arrest in NTERA2-R cells without influencing cell viability, whereas demethyloxyaaptamine and isoaaptamine potently induced apoptosis. Proteomic screening was applied to assess proteins altered by treatment with 1, 2 and 3. Treatment at the IC50 for 48 h resulted in an alteration of 16-22 proteins (Fig. 2). Selected targets were subjected to analysis with Western blotting. Changes in the 2D map frequently resulted from post-transcriptional modifications rather then from changes in total protein expression. For example, aaptamine treatment caused hypusination of eIF5A at lysin50 (activation of eIF5A), phosphorylation of cofilin-1, and dephosphorylation of vimentin. Demethyloxyaaptamine treatment resulted in up-regulation of Hsp70 and differential phosphorylation of isoforms of alpha-enolase. Isoaaptamine caused dissociation of the nuclear matrix-associated nucleolin complex. Interestingly, 2 and 3 did not induce the eIF5A hypusination. A hypothetical pathway analysis, performed by IPA network analysis, put the following factors in a central position of the network: myc and p53 for aaptamine; TNF for demethyloxyaaptamine; and all three for isoaaptamine, thus identifying potential targets or effectors of the substances. Download : Download full-size image Fig. 1 . Aaptamine (1), demethyl-oxyaaptamine (2) and isoaaptamine (3).

Time-Dependent Modulation of Mitogen Activated Protein Kinases and AKT in Rat Hippocampus and Cortex in the Pilocarpine Model of Epilepsy

The epileptogenesis may involve a variety of signaling events that culminate with synaptic reorganization. Mitogen-activated protein kinases (MAPKs) and AKT may be activated by diverse stimulus including neurotransmitter, oxidative stress, growth factors and cytokines and are involved in synaptic plasticity in the hippocampus and cerebral cortex. The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. We analyze the phosphorylation profile of MAPKs (ERK1/2, p38(MAPK), JNK1/2/3) and AKT by western blotting in the hippocampus (Hip) and cortex (Ctx) of male adult wistar rats in different periods, after pilocarpine induced status epilepticus (Pilo-SE) and compared with control animals. Biochemical analysis were done in the Hip and Ctx at 1, 3, 12h (acute period), 5days (latent period) and 50days (chronic period) after Pilo-SE onset. Hence, the main findings include increased phosphorylation of ERK1 and p38(MAPK) in the Hip and Ctx 1 and 12h after the Pilo-SE onset. The JNK2/3 isoform (54kDa) phosphorylation was decreased at 3h after the Pilo-SE onset and in the chronic period in the Hip and Ctx. The AKT phosphorylation increased only in the Hip during the latent period. Our study demonstrates, in a systematic manner, the profile of MAPKs and AKT modulation in the hippocampus and cerebral cortex in response to pilocarpine. Based in the role of each signaling enzyme is possible that these changes may be related, at least partially, to modifications in the intrinsic neuronal physiology and epileptogenic synaptic network that appears in the MTLE-HS.

Abstract 3605: Effects of antibody-mediated EGF-receptor inhibition on ERK 1/2 isoform phosphorylation in organoid cultures

Abstract Inhibition of the EGF-receptor activity has been widely integrated into clinical practice for different tumor entities, e.g. non small lung cancer and colorectal cancer. Besides the application of tyrosine kinase inhibitors, anti-EGF-receptor treatment is mainly based on the application of therapeutic antibodies. As a standard procedure, patients are tested for KRAS mutation and only those without mutation are selected for anti-EGF-receptor therapy. Although KRAS testing is used to guide colorectal cancer treatment, clear heterogeneity in responses to treatment has been observed. The molecular mechanisms responsible for this heterogenity are still not fully understood, In the present study, we used a preclinical model based on tumor tissue slices for a more detailed evaluation of antibody-mediated EGF-receptor inhibition. In previous experiments, the diffusion of antibodies into organoid cultures and the detection of their functional effects had been successfully shown. Tumor tissue slices from five colorectal cancer patients have been treated with an anti-EGF-receptor antibody for 48 hours. Using immunohistochemical staining, one patient showed a strong expression of the EGF-receptor, one was weak positive and three displayed a moderate staining. As readout of treatment effects, the expression and phosphorylation levels of selected key proteins from two EGF-receptor downstream pathways, the MAPK-pathway and Akt-pathway, were measured using the multiplex technology platform from Meso Scale Discovery. For a more detailed view, we have integrated the NanoPro technology in our pathway analysis approach. This new method enables the identification of distinct isoform phosphorylations and thus is able to display the complex regulation of phosphorylation patterns in detail. By using both methods, we observed a dose-dependent regulation of total- and phospho-ERK in the organoid cultures after anti-EGF-receptor treatment. Deeper analysis using the NanoPro technology showed that the regulation of isoform phosphorylation patterns of ERK1/2 differed among patients, representing the heterogeneity of individual responses among patients. In summary, the combination of the organoid drug testing platform and the NanoPro technology can potentially be useful to identify predictive phosphorylation patterns of tumor cells within their natural tumor microenvironment. This may facilitate the discovery of predictive biomarker for the response to anti-EGF-receptor treatment and further improve personalized medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3605. doi:1538-7445.AM2012-3605