Tyrosine Phosphorylation Profile Research Articles

Modulatory effect of MG-132 proteasomal inhibition on boar sperm motility during in vitro capacitation.

A series of biochemical and biophysical changes during sperm capacitation initiates various signaling pathways related to protein phosphorylation leading to sperm hyperactivation, simultaneously with the regulation of proteasomal activity responsible for protein degradation and turnover. Our study aimed to unveil the role of the proteasome in the regulation of boar sperm motility, hyperactivated status, tyrosine phosphorylation, and total protein ubiquitination. The proteolytic activity of the 20S proteasomal core was inhibited by MG-132 in concentrations of 10, 25, 50, and 100 μM; and monitored parameters were analyzed every hour during 3 h of in vitro capacitation (IVC). Sperm motility and kinematic parameters were analyzed by Computer Assisted Sperm Analysis (CASA) during IVC, showing a significant, negative, dose-dependent effect of MG-132 on total and progressive sperm motility (TMOT, PMOT, respectively). Furthermore, proteasomal inhibition by 50 and 100 μM MG-132 had a negative impact on velocity-based kinematic sperm parameters (VSL, VAP, and VCL). Parameters related to the progressivity of sperm movement (LIN, STR) and ALH were the most affected by the highest inhibitor concentration (100 μM). Cluster analysis revealed that the strongest proteasome-inhibiting treatment had a significant effect (p ≤ 0.05) on the hyperactivated sperm subpopulation. The flow cytometric viability results proved that reduced TMOT and PMOT were not caused by disruption of the integrity of the plasma membrane. Neither the protein tyrosine phosphorylation profile changes nor the accumulation of protein ubiquitination was observed during the course of capacitation under proteasome inhibition. In conclusion, inhibition of the proteasome reduced the ability of spermatozoa to undergo hyperactivation; however, there was no significant effect on the level of protein tyrosine phosphorylation and accumulation of ubiquitinated proteins. These effects might be due to the presence of compensatory mechanisms or the alteration of various ubiquitin-proteasome system-regulated pathways.

Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.

Separation of the CaV1.2‐CaV1.3 calcium channel duo prevents type 2 allergic airway inflammation

Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. We generated mice deleted for Cav 1.2 in T cells or Cav 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav 1.3has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.

24-epibrassinolide-induced growth promotion of wheat seedlings is associated with changes in the proteome and tyrosine phosphoproteome.

Brassinosteroids (BRs) represent a unique class of steroidal plant hormones that display pronounced growth-promoting activity at very low concentrations. Although many efforts have been made to characterize the molecular basis of BR action, little is known about the mechanisms behind the growth-promoting effect of BRs at protein level. Proteomic analysis of response to the steroid plant hormone 24-epibrassinolide (EBR) in wheat seedling shoots (Triticum aestivum L.) was performed using two-dimensional electrophoresis (2-DE) and immunoblotting with highly specific antibodies (PY20) to phosphotyrosine. EBR-modulated proteins and phosphotyrosine polypeptides were identified using MALDI-TOF mass spectrometry. The study revealed that EBR-stimulated growth of wheat seedlings was accompanied by changes in the content of multiple proteins as well as in tyrosine phosphorylation of numerous polypeptides. Among them, 22 differentially accumulated proteins and 13 phosphotyrosine proteins were identified. Based on their performed functions, the identified proteins are involved in physiological processes (photosynthesis, growth, energy and amino acid metabolism) closely associated with intensification of plant metabolism. The EBR-induced changes in protein abundance and tyrosine phosphorylation profile may contribute to growth stimulation of wheat seedlings under the action of EBR. The obtained data suggest an important role for EBR in the activation of protein metabolism underlying fundamental physiological processes, including growth promotion.

FGFR3 signaling and function in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target.MethodsMS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1–3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival.ResultsHigh FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare.ConclusionsThese results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion.AQe7iP6srQZx6BoH-5jRjTVideo abstract.

Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks.

The small-molecule drug NT157 has demonstrated promising efficacy in preclinical models of a number of different cancer types, reflecting activity against both cancer cells and the tumor microenvironment. Two known mechanisms of action are degradation of insulin receptor substrates (IRS)-1/2 and reduced Stat3 activation, although it is possible that others exist. To interrogate the effects of this drug on cell signaling pathways in an unbiased manner, we have undertaken mass spectrometry-based global tyrosine phosphorylation profiling of NT157-treated A375 melanoma cells. Bioinformatic analysis of the resulting dataset resolved 5 different clusters of tyrosine-phosphorylated peptides that differed in the directionality and timing of response to drug treatment over time. The receptor tyrosine kinase AXL exhibited a rapid decrease in phosphorylation in response to drug treatment, followed by proteasome-dependent degradation, identifying an additional potential target for NT157 action. However, NT157 treatment also resulted in increased activation of p38 MAPK α and γ, as well as the JNKs and specific Src family kinases. Importantly, cotreatment with the p38 MAPK inhibitor SB203580 attenuated the antiproliferative effect of NT157, while synergistic inhibition of cell proliferation was observed when NT157 was combined with a Src inhibitor. These findings provide novel insights into NT157 action on cancer cells and highlight how globally profiling the impact of a specific drug on cellular signaling networks can identify effective combination treatments. Mol Cancer Ther; 17(5); 931-42. ©2018 AACR.

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility.

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO-/- mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO-/- mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO-/- sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO-/- sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO-/- sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.

A simple toolset to identify endogenous post-translational modifications for a target protein: a snapshot of the EGFR signaling pathway.

Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role and studying its potential cross-talk with other PTMs is a challenging process. A set of highly sensitive tools termed as Signal-Seeker kits was developed, which enables rapid and simple detection of PTMs on any target protein. The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate, and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential cross-talk between PTMs. As a proof-of-concept experiment, the acetylation (Ac), tyrosine phosphorylation (pY), SUMOylation 2/3, and ubiquitination (Ub) profiles of the epidermal growth factor (EGF) receptor (EGFR)–Ras–c-Fos axis were examined in response to EGF stimulation. All ten previously identified PTMs of this signaling axis were confirmed using these tools, and it also identified Ac as a novel modification of c-Fos. This axis in the EGF/EGFR signaling pathway was chosen because it is a well-established signaling pathway with proteins localized in the membrane, cytoplasmic, and nuclear compartments that ranged in abundance from 4.18 × 108 (EGFR) to 1.35 × 104 (c-Fos) molecules per A431 cell. These tools enabled the identification of low abundance PTMs, such as c-Fos Ac, at 17 molecules per cell. These studies highlight how pervasive PTMs are, and how stimulants like EGF induce multiple PTM changes on downstream signaling axis. Identification of endogenous changes and potential cross-talk between multiple PTMs for a target protein or signaling axis will provide regulatory mechanistic insights to investigators.

Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling

Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a novel taxonomy for particular cancers, and provide insights into PDAC biology that can be exploited for improved patient management.

Abstract 3274: Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties

Abstract Background: Epidermal Growth Factor Receptor (EGFR) is a biologic target for cancer therapy. Clinical response to EGFR inhibitors is variable. Recently, EGFR mutations were found to predict response to anti-EGFR therapy. Since a few children with Neuroblastoma (NB) benefited from EGFR inhibitors in clinical trials, we screened for EGFR mutations in primary NB. Interestingly, we identified the expression of both EGFRvIII and a novel mutation, EGFRΔ768, in these tumors. In this study, we characterized the EGFRΔ768 mutation by comparing its biological and biochemical effect with that of wild type (WT)-EGFR and EGFRvIII. Hypothesis: EGFRΔ768 expression confers an aggressive cancer phenotype in NB cells Materials and Methods: We obtained 62 Snap Frozen primary neuroblastic tumors from the Columbus Bipathology Repository Center and 80 NB RNA from the Children Oncology Group (COG). Primary NB were prepared for RT-PCR and sequencing analyses using EGFR exon 1 & 8 primers. Five NB cell lines were obtained and confirmed by STR profiling. The EGFRΔ768 and EGFRvIII deletions were subcloned into a WT-EGFR expression vector with a C-terminal GFP tag. Next, stable 3T3 and NB clones expressing WT and mutant EGFR were generated. The clones were visualized under fluorescence microscopy. Invasion and XTT assays were performed with the stable clones. Responses to Erlotinib from 0.01 - 1 ng/ul and Etoposide from 0.5 - 2 uM were compared among the clones by XTT and western analyses. EGFR phosphorylation and downstream signaling pathways were examined by Western Blot and phosphokinase array. Results: We previously reported that 32% (20/62) of the Columbus neuroblastic tumors expressed EGFR extracellular deletion mutants, EGFRΔ768 (an in-frame deletion from nucleotides 102 of exon 2 to 869 of exon 7) and EGFRvIII (Δ801). In the 80 COG NB RNA, 7 expressed EGFRΔ768, 7 expressed EGFRvIII and 1 expressed both. EGFRΔ768 expression was also detected in the NB cell line, BE2M17. This novel mutant localized to the cell surface and enhanced the invasion and proliferation of NB cells in vitro when compared to control GFP+ cells. Its expression also confers sensitivity to Erlotinib, but the biochemical inhibition of Erlotinib on EGFRΔ768 is significantly less effective than that on activated WT-EGFR from 0.01 - 0.1 ng/ul. EGFRΔ768+ cells were also significantly more resistant to Etoposide than the GFP and WT-EGFR+ cells. Although both EGFRΔ768 and EGFRvIII autophosphorylated without ligand, the cellular proliferation rate and baseline autophosphorylation of EGFRΔ768+ cells were significantly higher than those of EGFRvIII+ cells. Finally, we found that EGFRΔ768 did not activate downstream signaling pathways of WT-EGFR such as MAPK or STAT. It has a different tyrosine phosphorylation profile than WT-EGFR. Conclusion: EGFRΔ768 confers aggressive cancer cell behaviors and has distinct biological as well as biochemical properties. Citation Format: James Keller, Kristen A. VanHeyst, Anjaruwee S. Nimnual, Mathew Varghese, Michael J. Hayman, Edward L. Chan. Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2015-3274

Abstract 2264: Dual effects of podoplanin on the regulation of cancer cell gene expression and platelet CLEC-2 signaling during cancer metastasis

Abstract Podoplanin (PDPN) is frequently up-regulated in squamous cell carcinoma and is a marker for poor prognosis. PDPN facilitates cancer metastasis and tumor cell-induced platelet aggregation (TCIPA) by modulating cancer cell cytoskeleton reorganization and platelet C-type lectin-like receptor 2 (CLEC-2) activation. In this study, the effects of PDPN on the regulation of cancer cell gene expression and platelet CLEC-2 signaling are investigated. Using xenograft injection of the C6 glioma cells into nude mice as a metastatic model, the cells that colonized at lung tissue were isolated to establish a subline C6-Lung that was found to express high levels of PDPN. Knockdown of PDPN expression in C6-Lung resulted in the abrogation of TCIPA, indicating that PDPN plays a key role in platelet activation and TCIPA. Microarray analyses revealed differential gene expression signatures between low and high PDPN-expressed cells. Several genes were up-regulated (Sncg, Timp1, Mmp3 and Pla1a) and down-regulated (Calcb, Filip1, Agrn and Itga4) significantly in high PDPN-expressed C6-Lung cells. On the other hand, distinct platelet protein tyrosine phosphorylation profiles were revealed when platelets were treated with different CLEC-2 agonists including PDPN, aggretin and fucoidan. In addition to the activation of common CLEC-2 signaling proteins such as Src family kinase, Syk, and phospholipase Cγ2, Akt1/PDK1 and PKCμ were identified as the two novel signaling pathways activated by PDPN, indicating the presence of SFK-independent pathway downstream of CLEC-2. Our data together provide a definitive evidence for the presence of a unique network for PDPN-regulated cancer cell gene expression and platelet CLEC-2 signaling that shed a new insight for the functional roles of PDPN in cancer metastasis. Citation Format: Ching-Ping Tseng, Yao-Wen Chang, Pei-Wen Hsieh, Ju-Chien Cheng. Dual effects of podoplanin on the regulation of cancer cell gene expression and platelet CLEC-2 signaling during cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2264. doi:10.1158/1538-7445.AM2015-2264

Targeted Cancer Therapy in High-Risk Pediatric Leukemia Using Global Phosphotyrosine Profiling

Introduction: While cure rates for children with acute lymphoblastic leukemia (ALL) are approaching 90% with conventional chemotherapeutic regimens, certain high-risk patient subsets such as early T-cell precursor ALL (ETP-ALL) and Philadelphia Chromosome-like (Ph-like) ALL have an aggressive disease profile and poor prognosis. More recently whole genome and transcriptome sequencing of these high-risk subtypes have revealed several activating gene fusions, alterations and mutations that could result in constitutively activated tyrosine kinases (TKs). Activated TKs are then capable of phosphorylating downstream substrates and impacting several key signaling pathways, resulting in increased cell survival, proliferation and differentiation. Further, the highly heterogeneous nature of these subtypes, along with activating fusions/mutations, makes them refractory to standard chemotherapy. Consequently, there is an urgent need to develop tailored therapeutic strategies for the treatment of these high-risk ALL subtypes. Recent advances in mass-spectrometry and the use of anti-phosphotyrosine antibodies for enrichment of tyrosine phosphorylated peptides have greatly facilitated characterization of the global tyrosine phosphorylation state in cancer cells and identified activated TKs that could be therapeutically targeted. Here we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL.Methods: In this study, we have established an MS-based phosphotyrosine profiling approach in patient derived xenografts (PDXs) of high-risk pediatric ALL patients and integrated it with a spike-in SILAC quantitative tool to identify and quantify dysregulated TK activity across 16 PDXs. We further extended our study on markedly altered tyrosine phosphorylation in 4 PDXs to assess the therapeutic potential of specific TK inhibitors (TKIs). Immunoblots were performed to validate activated sites and their dephosphorylation upon TKI treatment. RT-PCR and Exome sequencing was carried out to detect novel fusion partners and point mutation sites to validate the activated TK profiles in these PDXs. In vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. PDXs were established from ETP-ALL, Ph-like ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice. Engraftment and in vivo drug responses were assessed by enumeration of the proportion of human versus mouse CD45+cells in the murine PB.Results: Using a quantitative phosphotyrosine profiling method in 16 PDXs, we mapped close to 1900 class I phosphosites with >0.75 localization probability and 99% confidence, of which 1394 tyrosine phosphorylated sites had a heavy SILAC partner that allowed quantification. Such profiling could accurately classify the leukemias into either T or B-cell lineages with the high-risk ETP and Ph-like ALL clustering as a distinct group. In particular, PDXs with activated tyrosine phosphorylation profiles of ABL1, FLT3 and JAK were targeted with commercially available TKIs both in vitro and in vivo. Subsequent analysis to investigate the aberrant ABL1 and FLT3 signaling showed a NUP214-ABL1 translocation unique to BCP-ALL in one PDX, and a novel Y572S FLT3 mutation in another. Importantly, using a pre-clinical in vivo xenograft model, the activated JAK-STAT signaling observed in one ETP-ALL PDX was targeted with the JAK1/2 inhibitor, ruxolitinib, leading to a significant decrease in the leukemic blast population in the murine PB. Aberrant ABL1 kinase signaling indicated dasatinib treatment in a Ph+-ALL PDX and a PDX with high phospho-ABL1 (harboring the NUP214-ABL1 translocation), and a complete response and significant progression delay, respectively, were achieved in vivo. Similarly, the uniquely activated FLT3 in one PDX (Y572S mutation) correlated with an in vivoobjective response to the multi-kinase inhibitor sunitinib.Conclusions: This study demonstrates the direct application of an unbiased and quantitative tool to identify aberrant TK signaling in high-risk ALL PDXs and highlights its potential to identify tractable drug targets.This research was supported by NCI NO1CM42216 and by the Australian National Health and Medical Research Council. DisclosuresNo relevant conflicts of interest to declare.

Mammary Tumor Formation and Metastasis Evoked by a HER2 Splice Variant

The HER2 gene is amplified and overexpressed in approximately 20% of invasive breast cancers where it is associated with metastasis and poor prognosis. Here, we describe a constitutively active splice variant of HER2 (Delta-HER2) in human mammary epithelial cells that evokes aggressive breast cancer phenotypes. Delta-HER2 overexpression in mammary epithelial cells was sufficient to reduce apoptosis, increase proliferation, and induce expression of mesenchymal markers, features that were associated with greater invasive potential in three-dimensional cultures in vitro and more aggressive tumorigenicity and metastasis in vivo. In contrast, overexpression of wild-type HER2 was insufficient at evoking such effects. Unbiased protein-tyrosine phosphorylation profiling in Delta-HER2-expressing cells revealed increased phosphorylation of several signaling proteins not previously known to be controlled by the HER2 pathway. Furthermore, microarray expression analysis revealed activation of genes known to be highly expressed in ER-negative, high-grade, and metastatic primary breast tumors. Together, our results provide mechanistic insights into the activity of a highly pathogenic splice variant of HER2.

Identification of signaling molecules responsible for IL-2-mediated cytokine expression in human T cells (P6343)

Abstract IL-2 is an important cytokine regulating growth, differentiation and function of T cells. IL-2 directly induces production of several Th2 cytokines by T cells, especially of human origin. The mechanisms of IL-2-mediated cytokine synthesis have not been clarified, though the contribution of IL-2 receptor (IL-2R)β has been suggested. Here, by employing a proteomic approach, the intracellular signaling molecules downstream of IL-2Rβ were investigated. IL-2Rα and IL-2Rγ were stably transfected in a T cell hybridoma established by fusing an antigen-specific human T cell clone and BUC cells. The resulting hybridoma was further transfected with full-length and intracellular component-truncated IL-2Rβ. Then, the differential protein tyrosine phosphorylation profiles in those cells following IL-2 stimulation were examined by two-dimensional gel electrophoresis and mass spectrometry. Several proteins specifically phosphorylated in full-length IL-2Rβ-expressing cells were identified. Among them, overexpression of protein phosphatase (PP)1β significantly augmented IL-2-induced IL-5 and IL-13 expression in human peripheral CD4+ T cells, whereas TCR-induced cytokine expression was not affected. In contrast, introduction of PP1 regulatory subunit 2 as well as treatment with tautomycin, a PP1 inhibitor, but not ocadaic acid, a PP2A inhibitor, inhibited the IL-2-mediated responses. Conclusively, PP1 is a key molecule responsible for IL-2-mediated cytokine synthesis in human T cells.

Molecular Dissection of AKT Activation in Lung Cancer Cell Lines

AKT is a critical signaling node downstream of phosphoinositide 3-kinase (PI3K), which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung carcinoma cell lines under serum starvation conditions. We identified 13 lines, which showed persistent AKT activation in the absence of serum. In 12 of 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGF receptor (EGFR) or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTOR complex 2 (mTORC2)-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Furthermore, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Overexpression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features.

Imatinib-dependent tyrosine phosphorylation profiling of Bcr-Abl-positive chronic myeloid leukemia cells

Fondation Leducq: the Alliance for CamKII signaling in heart 128 disease (CP, AS, AJRH), and the Netherlands Proteomics Centre, embedded in the 129 Netherlands Genomics Initiative, (CP, AS, AJRH) and Science Foundation Ireland under 130 Grant No. 06/CE/B1129

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Galectin-3 secretion and tyrosine phosphorylation is dependent on the calpain small subunit, Calpain 4

Cell adhesion and migration are important events that occur during embryonic development, immune surveillance, wound healing and in tumor metastasis. It is a multi-step process that involves both mechanical and biochemical signaling that results in cell protrusion, adhesion, contraction and retraction. Each of these events generates mechanical forces into the environment measured as traction forces. We have previously found that the calpain small subunit, Calpain 4, is required for normal traction forces, and that this mechanism is independent of the catalytic activities of the holoenzymes that are formed between Calpain 4 and each of the proteolytic heavy chains of Calpain 1 and 2. To define a potential mechanism for the Calpain 4 regulation of traction force, we have evaluated the levels of tyrosine phosphorylation, a hallmark of force dependent signaling within focal adhesions. Using 2D gel electrophoresis we compared tyrosine phosphorylation profiles of Calpain 4 deficient mouse embryonic fibroblasts (MEFs) to the levels in wildtype MEFs and MEF’s deficient in the large catalytic subunits, Capn1 and Capn2. Of particular interest, was the identification of Galectin-3, a galactose binding protein known to interact with integrins. Galectin-3 has previously been shown to regulate cell adhesion and migration in both normal and tumor cells; however its full mechanism remains elusive. We have found that Calpain 4 is essential for the tyrosine phosphorylation of galectin-3, and its ultimate secretion from the cell, and speculate that its secretion interferes with the production of traction forces.

Tyrosine Phosphorylation Profiling in FGF-2 Stimulated Human Embryonic Stem Cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup.