Basal Tyrosine Phosphorylation Research Articles

Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors.

Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9mg/kg dasatinib, with maximal reduction observed 2h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches.

Preclinical evaluation of LCK as a novel therapeutic target in YAP-activated and FGFR2-altered cholangiocarcinoma.

463 Background: Molecularly targeted therapy is gaining traction in cholangiocarcinoma with the first FDA approval of a targeted agent in 2020, the FGFR inhibitor pemigatinib. Yet response rates and efficacy are modest, and most cholangiocarcinoma patients still lack targeted therapy options. The hippo pathway effector, YAP, is an oncogene that is activated in the majority of cholangiocarcinoma, yet attempts to develop YAP inhibitors have not yet been clinically successful. Recently, we described a novel mechanism of YAP regulation, via tyrosine phosphorylation by the Src-family kinase LCK. Therefore we hypothesized that LCK inhibition by may be therapeutic in cholangiocarcinoma through suppression of YAP activity. Methods: NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. In vitro pharmacodynamics were defined. In cholangiocarcinoma cells, specificity for LCK inhibition was determined by mass spectrometry-based unbiased analysis of the tyrosine phosphoproteome following treatment with NTRC 0652-0 or genetic deletion of LCK. YAP phosphorylation and cotranscriptional activity, cell viability, and apoptosis were assessed. A panel of eight cholangiocarcinoma patient derived organoids (PDO) were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft (PDX) models bearing FGFR2-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. Results: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to YAP inhibition and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression. PDOs demonstrated variable sensitivity to NTRC 0652-0, correlated with basal YAP tyrosine phosphorylation and drug-induced suppression of YAP co-transcriptional activity. FGFR2-altered cholangiocarcinoma was also identified as a subset with enrichment of YAP target genes. Cells from an FGFR2-altered PDX were sensitive to NTRC 0652-0 despite being primarily resistant to pemigatinib. In two PDX models of FGFR2-altered cholangiocarcinoma, daily oral treatment with NTRC 0652-0 inhibited YAP tyrosine phosphorylation, and resulted in stable plasma and tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Conclusions: A novel LCK inhibitor, NTRC 0652-0, reduced YAP signaling and demonstrated preclinical efficacy in multiple patient-derived models of cholangiocarcinoma. LCK is a novel therapeutic target in cholangiocarcinoma, and YAP activation or FGFR2-alteration are potential biomarkers for response.

Role of Src‐Dependent Phosphorylation of Mitofusin 2 in Endoplasmic Reticulum‐Mitochondria Tethering

Introduction : Contact sites between the mitochondria and endoplasmic reticulum (ER) are one of the key structures involved in regulating lipid, Ca2+ and reactive oxygen species exchange across the two organelles which is critical for the maintenance of mitochondrial bioenergetics. Recent studies have reported that a major component of the tethering structures between the two organelles is mitofusin 2 (Mfn2), a GTPase associated with fusion of the outer mitochondrial membrane (OMM) as well as the activities of mitophagy and ER stress. While several post-translational modifications (PTMs) that influence the fusion and mitophagy functions of Mfn2 have been identified, it remains unclear whether there are PTMs that can regulate its tethering function. Data from mass spectroscopy reveals that there is basal tyrosine phosphorylation (P-Tyr) of Mfn2, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases (proline rich tyrosine kinase 2 [Pyk2] and proto-oncogene Src kinase [Src]). Objective : To determine whether P-Tyr of Mfn2 regulates Mfn2 functions. Methods HEK293T cells stably overexpressing shRNA targeted to a specific negative regulator of Src, C-terminal Src kinase (CSK) or a control vector were subjected to several biochemical (Fluorescent western blotting), cell biological (Transmission electron microscopy [TEM], Förster Resonance Energy Transfer [FRET] between cyan-fluorescent protein targeted to the OMM and yellow-fluorescent protein targeted to the ER membrane observed under confocal microscopy) and physiological (mitochondrial calcium [mtCa2+] uptake measurements using a mitochondria-targeted Ca2+ biosensor, mt-RCaMp1h) assays. Results Overexpression of Pyk2 and Src lead to P-Tyr of Mfn2 with Src having greater P-Tyr levels of Mfn2. Generation of a HEK293T cell line with stable knockdown of CSK reveals that CSK knockdown promotes Src activation in the mitochondria and increases P-Tyr of Mfn2 without changing expression levels of Src and Mfn2. CSK knockdown (CSK-KD) cells showed decreased distance between the ER and OMM as assessed by FRET measurement under confocal microscopy. This change was abolished by pretreatment with a Src-specific inhibitor, PP2. TEM images also showed that CSK knockdown decreases the distance between OMM and ER without changing the length of interface. To assess the impact of changes in the physical interaction between ER and mitochondria, mtCa2+ uptake profiles in CSK-KD cells were observed. In CSK-KD cells, mtCa2+ uptake in response to cytosolic Ca2+ elevation induce by Gq-protein coupled receptor stimulation was enhanced with faster kinetics compared to that in control cells. While physical coupling between ER and mitochondria increased, we found that hallmarks of ER stress and mitophagy were not altered in CSK-KD cells compared to control cells. Conclusion : Src-dependent phosphorylation of Mfn2 decreased the distance between the OMM and ER and lead to increased Ca2+ exchange between ER and mitochondria.

Anethole compromises human sperm function by affecting the sperm intracellular calcium concentration and tyrosine phosphorylation

Anethole is a natural anisole derivative that has been widely used in food and daily chemical industries, agricultural applications and the traditional medicine. It is closely related to aspects of daily life, and humans can easily be exposed to it. Although the reproductive toxicity of anethole was shown in the rat, its effect on human reproduction remains unknown. In this study, we examined the effect of anethole on human sperm in vitro. Different anethole doses (0.1, 1, 10, and 100 μM) were applied to ejaculated human sperm. Fertilization-essential functions, as well as the intracellular calcium concentration ([Ca2+]i) and tyrosine phosphorylation, two vital factors for regulating sperm function, were measured. The results indicated that 10 and 100 μM anethole significantly reduced the motility, hyperactivation, and penetration ability of human sperm (P < 0.05) and inhibited the increase in human sperm functions induced by progesterone, a hormone essential for sperm function activation. Additionally, 10 and 100 μM anethole decreased both basal and progesterone-increased tyrosine phosphorylation, [Ca2+]i, and the current of CATSPER, a cation channel of sperm predominant for Ca2+ influx. These results suggest that anethole inhibits human sperm functions by reducing sperm [Ca2+]i through CATSPER and suppressing tyrosine phosphorylation in vitro, raising the fact that the caution is needed when overtaking anethole.

Abstract P2-06-03: Insulin receptor isoform signaling in breast cancer

Abstract Recent data shows that insulin receptor (IR) and the type I insulin-like growth factor receptor (IGF1R) play important roles in breast cancer cell biology. Targeting only IGF1R has not been successful perhaps due to compensation by IR. IR exists in two isoforms, fetal isoform IR-A is a splice variant of IR which excludes exon 11. The adult/metabolic isoform IR-B is the predominant species expressed in normal tissues, while the fetal form IR-A is more highly expressed in breast cancer. IR-A mRNA in endocrine resistant cells is expressed at levels 24-fold higher than IGF1R expression (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). In addition to its homodimer, IR-A can also dimerize with IGF1R to form a hybrid. Homodimer IR-A responds to IGF-II and insulin, while IGF1R/IR-A hybrid can also respond to IGF-I. Previous studies in our lab showed down regulation of IGFIR increases the sensitivity of breast cancer cells to insulin. To further investigate the roles of IR-A and IR-B in breast cancer biology, we cloned IR-A and IR-B isoforms into pLV-mCherry and pLJM1EGFP lentiviral expression vectors. Then MCF-7L breast cancer cells were infected with IR-A, IR-A vector control, IR-B and IR-B vector control. Both pooled and single clones were studied. Our results showed that both IR-A and IR-B were highly expressed in MCF-7L cells with the introduced IR tagged species present at a higher molecular weight confirmed by immunoprecipitation and immunoblot. The lower migrating species was endogenous IR. IR-A-pool/Clones had strong basal IR tyrosine phosphorylation, while IR-B-pool/clones did not. This basal phosphorylation did not activate downstream signaling as measured by pAKT and pErk1,2. IR-A basal phosphorylation was completely inhibited by BMS754807 (0.3μM), a dual IGFIR/IR tyrosine kinase inhibitor. Total levels of IRS1, IRS2, IGF1R, ERa, AKT and Erk1,2 levels were not changed in the over-expressing cells. Ligands-IGF-I (5nM), IGF-II (10nM) and Insulin (10nM) stimulated IGF1R/IR, IRS, AKT and Erk1,2 phosphorylation in IR-A-pool and high expression clone IR-A-G5. Cells expressing IR-A activated downstream signaling (IRS, AKT and Erk1,2) at 0.1nM insulin, while IR-A vector control cells required 1nM insulin. IR-B-pool overexpressing cells were not more sensitive to insulin compared to parental cells. Insulin significantly increased Erk1,2 phosphorylation in IR-A-G5 cell while IGF-I stimulation was minimal. In contrast, Erk1,2 phosphorylation in parental cells and vector control cells was primarily mediated by IGF-I, not insulin. IR-A overexpressing cells were stimulated in monolayer growth by insulin. These data show that IR-A expression, as seen in endocrine resistant breast cancer cells, sensitizes breast cancers to low concentrations of insulin. Thus, IR-A expression could serve as a target in breast cancer. Citation Format: Zhang X, Chan JY, Pan Y, Dong C, Yee D. Insulin receptor isoform signaling in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-03.

MicroRNA203a suppresses glioma tumorigenesis through an ATM-dependent interferon response pathway.

Glioblastoma (GBM) is a deadly and incurable brain tumor. Although microRNAs (miRNAs) play critical roles in regulating the cancer cell phenotype, the underlying mechanisms of how they regulate tumorigenesis are incompletely understood. We previously showed that miR-203a is expressed at relatively low levels in GBM patients, and ectopic miR-203a expression in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by interferon (IFN) or temozolomide in vitro, and inhibited GBM tumorigenesis in vivo. Here we show that ectopic expression of miR-203a in GBM cell lines promotes the IFN response pathway as evidenced by increased IFN production and IFN-stimulated gene (ISG) expression, and high basal tyrosine phosphorylation of multiple STAT proteins. Importantly, we identified that miR-203a directly suppressed the protein levels of ataxia-telangiectasia mutated (ATM) kinase that negatively regulates IFN production. We found that high ATM expression in GBM correlates with poor patient survival and that ATM expression is inversely correlated with miR-203a expression. Knockout of ATM expression and inhibition of ATM function in GBM cell lines inhibited cell proliferation and migration, increased sensitivity to apoptosis induced by therapeutic agents in vitro, and markedly suppressed GBM tumor growth and promoted animal survival. In contrast, restoring ATM levels in GBM cells ectopically expressing miR-203a increased tumorigenicity and decreased animal survival. Our study suggests that low miR-203a expression in GBM suppresses the interferon response through an ATM-dependent pathway.

Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Dictates Mitochondrial Ca2+ Overload and Cardiomyocyte Death

IntroductionMitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is critical for the cardiomyocyte survival and death. Basal tyrosine phosphorylation (P‐Y) of MCU was reported from mass spectroscopy of human tissues. However, it is still unclear whether the post‐translational modifications of the MCU (e.g. phosphorylation) modulates its channel properties as well as mitochondrial and cellular functions in cardiomyocytes.HypothesisP‐Y of MCU activates mitochondrial Ca2+ uptake and induces mitochondrial Ca2+ overload and cardiomyocyte death.MethodsNon‐phospho‐mimetic MCUs (MCU‐YFs; changed from Y to F) were generated by PCR‐based site direct mutagenesis. Mitochondrial Ca2+ concentration was measured by matrix‐targeted Ca2+ biosensors using confocal microscopy.Resultsa1‐adrenoceptor (a1‐AR) signaling activated mitochondrial matrix‐localized proline‐rich tyrosine kinase 2 (Pyk2) and enhanced the interaction between Pyk2 and MCU, which subsequently increased P‐Y of MCU and mitochondrial Ca2+ uptake in rat cardiomyocytes. We confirmed that two tyrosine sites showed an increase of phosphorylation levels in response to a1‐AR stimulation in situ (Fig. A). In addition, the overexpression of these MCU‐YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation, whereas wild‐type MCU overexpression dramatically accelerated mitochondrial Ca2+ uptake compared to control cells (Fig. B). Moreover, persistent a1‐AR stimulation increased mitochondrial reactive oxygen species (mROS) generation, cytochrome C release, and cardiomyocyte death. These effects were abolished by the overexpression of a dominant‐negative mutant of MCU in cardiomyocytes.ConclusionMCU contains Pyk2‐specific phosphorylation site(s) and P‐Y of MCU activates its channel function. Persistent P‐Y of MCU induces mitochondrial Ca2+ overload, mROS generation and apoptosis in cardiomyocytes.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

Pyk2‐Dependent Phosphorylation of Mitochondrial Ca 2+ Uniporter Modulates Mitochondrial Ca 2+ Uptake

The Ca2+ influx into mitochondrial matrix is primarily mediated via the mitochondrial calcium uniporter (MCU). Basal tyrosine phosphorylation (P-Y) of MCU was shown in mass spectroscopy of human tissues, but the functional relevance of MCU post-translational modifications including P-Y is still unknown. Here we show that adrenergic signaling activates mitochondria matrix-localized proline-rich tyrosine kinase 2 (Pyk2), and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent P-Y of MCUin cardiac cells.MCU contains 15 tyrosine residues and the phosphorylation prediction programs showed only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) as potential phosphorylation candidate sites for tyrosine kinases. These tyrosine residues were mutated into phenylalanine and non-phosphorylation mimetic MCU mutants (MCU-YFs) were stably expressed in HEK 293T cells. We found that only two tyrosine sites were phosphorylated in response to adrenergic stimulation in situ. In addition, overexpression of these MCU-YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation, whereas wild-type MCU transfection dramatically accelerates mitochondrial Ca2+ uptake compared to non-transfected cells. We conclude that MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent P-Y of MCU can modulate its channel functions and regulate mitochondrial Ca2+ uptake.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.

Cardiac α1‐adrenergic signaling accelerates mitochondrial Ca2+ uptake, ROS generation and cell death signaling through Pyk2‐dependent phosphorylation of the mitochondrial Ca2+ uniporter (893.9)

Mitochondrial Ca2+ homeostasis controls the balance of cell survival and death. The molecular identity of the mitochondrial Ca2+ uniporter (MCU) has been just reported and basal tyrosine phosphorylation of MCU was shown in mass spectroscopy of human tissues. However, the physiological and pathophysiological relevance of MCU post‐translational modifications are unknown. Here we reported that α1‐adrenoceptor (α1‐AR) signaling induces mitochondrial Ca2+ overload and reactive oxygen species (ROS) generation and activates cell death signaling through MCU tyrosine phosphorylation in cardiomyocytes. α1‐AR signaling activates mitochondria‐localized proline‐rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca2+ uptake via Pyk2‐dependent MCU phosphorylation which enhances tetrameric MCU channel pore formation. Moreover, mitochondrial Ca2+‐overload thorough this signaling stimulates mitochondrial superoxide generation, permeability transition pore (mPTP) activity, and initiates apoptotic cell death in adult cardiomyocytes. Thus, α1‐AR‐Pyk2‐MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca2+ overload, oxidative stress and cardiac injury under persistent adrenergic stimulation such as during heart failure.

Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

Activation of Hepatic Inflammatory Pathways by Catecholamines Is Associated With Hepatic Insulin Resistance in Male Ischemic Stroke Rats

Patients who experience acute ischemic stroke may develop hyperglycemia, even in the absence of diabetes. In the current study we determined the effects of acute stroke on hepatic insulin signaling, TNF-α expression, endoplasmic reticulum (ER) stress, the activities of c-Jun N-terminal kinase (JNK), inhibitor κB kinase β (IKK-β), and nuclear factor-κB (NF-κB) pathways. Rats with cerebral ischemia developed higher blood glucose, and insulin levels, and insulin resistance index, as well as hepatic gluconeogenic enzyme expression compared with the sham-treated group. The hepatic TNF-α mRNA and protein levels were elevated in stroke rats in association with increased ER stress, phosphorylation of JNK1/2 and IKK-β proteins, IκB/NF-κB signaling, and phosphorylation of insulin receptor-1 (IRS-1) at serine residue. The basal and insulin-stimulated tyrosine phosphorylation of IRS-1 and AKT proteins was reduced. In addition, acute stroke increased circulating catecholamines in association with hepatic adrenergic signaling activation. After administration of a nonselective β-adrenergic receptor blocker (propranolol) before induction of cerebral ischemic injury, hepatic adrenergic transduction, TNF-α expression, ER stress, and the activation of the JNK1/2, IKK-β, and NF-κB pathways, and serine phosphorylation of IRS-1 were all attenuated. In contrast, the phosphorylated IRS-1 at tyrosine site and AKT levels were partially restored with improved poststroke hyperglycemia and insulin resistance index. These results suggest that acute ischemic stroke can activate proinflammatory pathways in the liver by the catecholamines and is associated with the development of hepatic insulin resistance.

Abstract 18531: Adrenergic Stimulation Enhances Mitochondrial Ca 2 + Uptake and Cell Death Signaling Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca 2 + Uniporter

Introduction: Mitochondrial Ca 2+ homeostasis is crucial for balancing cell survival and death. The recent breakthrough discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study the role of mitochondrial Ca 2+ regulation in cardiac cells. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca 2+ entry through post-translational modifications of MCU are completely unknown. Hypothesis: Persistent adrenergic signaling induces mitochondrial Ca 2+ overload and activates cell death signaling through MCU tyrosine phosphorylation. Methods: Tyrosine kinase activation and tyrosine phosphorylation of MCU were observed in rat neonatal cardiomyocytes and H9C2 cardiac myoblasts. Mitochondrial Ca 2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca 2+ -sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP, using confocal microscopy. Results: α 1 -adrenoceptor (α 1 -AR) signaling activates mitochondria-localized Ca 2+ -dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, persistent α 1 -AR stimulation increases ROS production, mPTP activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Moreover, pretreatment of a potent Pyk2 inhibitor PF-431396 or overexpression of dominant-negative mutant of MCU effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: The α 1 -AR-Pyk2 dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca 2+ entry and apoptosis in cardiac cells. Pyk2-MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca 2+ overload, oxidative stress and cardiac injury under pathophysiological states such as heart failure, where catecholamine levels highly increase.

Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

Adrenergic Stimulation Accelerates Mitochondrial Ca2+ uptake by PYK2-Dependent Phosphorylation of Mitochondrial Ca2+ Uniporter in Cardiac H9C2 Cells

Background: Recent break-through discovery in the molecular identity of mitochondrial Ca2+ uniporter (MCU) opens the new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported in human sample mass spectroscopy, but the post translational modifications of MCU are completely unknown.Hypothesis: Tyrosine phosphorylation of MCU can modulate the mitochondrial Ca2+-uptake rate in cardiac cells.Methods: MCU was transiently or stably overexpressed in cardiac H9C2 cells. Tyrosine phosphorylation of MCU was detected by a general anti-phospho-tyrosine antibody. Mitochondrial Ca2+ concentration ([Ca2+]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam).Results: α1-adrenergic stimulation by phenylephrine enhanced the translocation of a Ca2+-dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) from cytosol to mitochondria followed by the increase in mitochondrial Pyk2 activity. Overexpressed MCU was exclusively localized at mitochondria and tyrosine residues in MCU were phosphorylated after Pyk2 activation. In addition, Pyk2 was bound to MCU at the basal condition and this interaction was enhanced by phenylephrine treatment. Moreover, Pyk2-dependent phosphorylation of MCU enhances MCU olgomerization observed by a conventional native PAGE. These effects were abolished by the co-transfection of kinase-dead Pyk2. In MCU-overexpressed cells, [Ca2+]m increased rapidly and reached to higher levels in response to cytosolic Ca2+ transients evoked by thapsigargin compared to non-transfected cells. Moreover, peak [Ca2+]m in MCU-overexpressed cells reached to much higher levels by phenylephrine pretreatment compared to non-treated cells.Conclusion: α1-adrenergic stimulation accelerates mitochondrial Ca2+ uptake through Pyk2-dependent direct phospholylation of MCU, which promotes the formation of tetrametric MCU channel pore. Our findings open up an exciting opportunity for investigating the first candidate cell signaling pathway for the MCU post translational modifications in cardiac cells.

Targeting the Cellular and Molecular Components of CD28 Mediated Survival Signaling in Multiple Myeloma

Abstract 722Very little is known about the cellular or molecular nature of pro-survival interactions between multiple myeloma (MM) and their bone marrow stromal hosts. CD28, the prototypical co-stimulatory molecule on T cells is overexpressed on MM relative to their normal counterparts. Clinical studies have independently shown that expression of CD28 or its ligand CD86 on myeloma cells portends poor survival in patients. We and others have shown that CD28 activation increases MM survival via NFkB accompanied by increased PI3K activity. Previously (ASH2010 and 2011), and since published, we have shown that myeloid dendritic cells (DC) form a vital part of the myeloma microenvironment, and myeloma cells interact with DC via a CD28-CD80/CD86 (B7) interaction. On the myeloma side, this interaction drives a CD28 mediated pro-survival signal, while on the DC side, it drives a B7 mediated induction of growth and immunosuppressive factors that modulate the MM survival niches in the bone marrow. In this work, we delineated key molecules downstream of CD28 that transduce the pro survival signal on the myeloma side as well as explored agents and strategies to target DC that help MM survive in the microenvironment. We also studied the role of DC IL-6 for MM survival in cocultures.We have previously shown that CD28 activation by anti-CD28 mAb involves activation of the multifunctional pro-survival transcription factor NFkB accompanied by increased PI3K activity. Through immunoassays and radioactivity assays, using myeloma cell lines that were transfected with wild type murine CD28 (mCD28 differs only by 3 amino acids from human hCD28) and mutants for PI3K binding site YMNM, we show that the CD28 ligation with anti-mCD28 antibodies induces immediate tyrosine phosphorylation of mCD28 (just as observed by others in T cells), followed by binding of p85, the catalytic subunit of PI3K and activation of VAV1 (Fig 1). Association of p85 and VAV1 activation depended on an intact PI3K binding site on CD28's cytoplasmic tail. Our studies on how this signal is transduced show no involvement of GRB2 or SLP76 more traditionally associated with CD28 signaling. Moreover, silencing SLP76 using siRNA did not affect antibody mediated protection of MM cells against serum starvation. Activation of human CD28 with anti-CD28 mAb also up-regulated the basal tyrosine phosphorylation of human CD28 further just as observed in murine CD28.Through EMSAs, we also show that similar to what we have previously seen with activating antibodies, increase in NFkB activity was observed in myeloma cells when they were cocultured with DCs while being exposed to melphalan, but this spike did not dissipate in the presence of B7 blocking reagents such as CTLA4Ig (Abatacept®) suggesting a low threshold of CD28 activation. Next we tried using agents that have been known to modulate microenvironment such as lenalidomide, a novel drug that is currently used in combination with other chemotherapeutic agents such as dexamethasone and Bortezomib (Velcade®). Survival assays using flow cytometry, show that lenalidomide treated DCs provided significantly lower protection against cell death compared to wild type DC. Lenalidomide DCs also show lower levels of B7 receptors which might be a contributing factor to this phenomenon. Blocking CD28-B7 interaction with CTLA4Ig or CD28(Fab) molecules reduced the pro-survival effect of DC on MM against dexamethasone (Fig 2) melphalan or bortezomib. In order to determine the role of IL-6 in an environment where myeloma cells derive pro-survival signals from their interaction with DC, we used neutralizing antibodies against human IL-6 in cocultures of MM cells with DC. Surprisingly, IL-6 neutralization did not affect the protective effect of DC on MM in cocultures in the presence of chemotherapeutic drugs like melphalan or dexamethasone (Fig 2).All these suggest that targeting DC might be a viable strategy towards augmenting chemotherapy, either by using agents that attenuate DC differentiation or agents that can block the CD28-B7 interaction between DC and MM. Moreover, MM cells do not seem to depend much on IL-6 as a survival factor when they are in contact with DC and its pro-survival effect is more visible when myeloma cells have to survive outside the microenvironment without DC (previous presentation at ASH 2011). [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract PR-7: Antitumor activity of the immunomodulatory peptide SCV-07 correlates with changes in macrophage STAT3 signal transduction and cytokine secretion.

Abstract The synthetic dipeptide gamma-D-glutamyl-L-tryptophan (SCV-07) is able to enhance immune responses in preclinical anti-viral and vaccine models and is currently under clinical evaluation for attenuation of radiation-induced oral mucositis in HNSCC patients. We recently demonstrated that SCV-07 also has anti-tumor efficacy in various syngeneic and nude mouse xenograft models despite lack of direct inhibition of the tumor cell growth in standard proliferation assays. Based on these data, along with observations that SCV-07 enhances a Th1-type immune response in vivo and binds with high affinity to cells and membranes from mouse thymocytes and macrophages, we sought to explore the hypothesis that SCV-07 has anti-tumor activity through modulation of immune cells in the tumor microenvironment. Towards this end we evaluated effects of SCV-07 on signal transduction and biological properties of T cells and macrophages in cell culture and in mouse tumor models. Using phosphoprotein detection arrays broad changes in global phosphorylation patterns were observed upon SCV-07 treatment of Jurkat T cells including STATs 3, 5 and 6, SHP2, several Src family kinases and various cytoplasmic and membrane proteins. We subsequently showed that SCV-07 leads to inhibition of cytokine-induced but not basal STAT3 tyrosine phosphorylation (PY) in Jurkat T cells and this required tyrosine phosphatase activity. New data using phosphospecific antibodies in western blots of Jurkat cells demonstrate that SCV-07 mediates an increase in basal and cytokine-induced phosphorylation of SHP2, a tyrosine phosphatase known to dephosphorylate STAT3. The phosphorylated residue was identified as Tyr542, known to activate SHP2. New findings also demonstrate that SCV-07 inhibits IL-6 stimulated STAT3 PY and induces SHP2 Tyr542 phosphorylation in primary mouse peritoneal macrophages treated in culture, similar to the findings with cell lines. Further evaluation of human PBMC showed that SCV-07 inhibited IL-6, IL-10 and IFNα induced STAT3 PY in monocytes from 2 of 3 PBMC donors. In a syngeneic B16 melanoma model we initially determined that SCV-07 had anti-tumor efficacy and this was accompanied by a decrease in serum CCL2, a cytokine produced by and chemotactic for monocytes/macrophages and which has its expression regulated by STAT3. New results show that SCV-07 can directly inhibit basal and cytokine induced CCL2 secretion from purified primary mouse peritoneal macrophages. Experiments are underway to evaluate the activation state and distribution of tumor infiltrated macrophages and T cells in B16 melanoma xenografts undergoing efficacy response to SCV-07 treatment and these findings will be presented. Taken together our data support a model whereby SCV-07 could mediate an anti-tumor effect by reducing both chemotaxis of tumor-growth promoting macrophages and secretion of tumor growth promoting cytokines in the tumor microenvironment, and also promote a shift from Th2 to Th1 profile of local T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-7.

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant (32)P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.