Major Tyrosine Kinase Research Articles

Abstract 14954: Mitochondrial C-Src Regulates Impaired Mitochondrial Bioenergetics in Right Ventricles in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) has poor prognosis with presence of right ventricular (RV) dysfunction. However, no RV targeted therapies are available. Mitochondrial (Mito) energy deficiency is detrimental on RV function in PH, but the underlying mechanism remains unclear. Tyrosine kinase phosphorylation (P-Tyr) has been a critical regulatory mechanism in mito. While mito localized c-Src is recognized as the major tyrosine kinase, little is known regarding its role in RV in PH. Hypothesis: We hypothesized that mito c-Src regulates impaired mito bioenergetics in RV in PH. Methods: PH was induced in adult Sprague-Dawley (SD) rats by injecting 20 mg/kg SU5416, followed by 3 wks of hypoxia (10% FiO 2 ) and then 4 wks of normoxia exposure. Control rats injected with vehicle were kept in normoxia. After echocardiography and hemodynamics, RV tissues were collected for mito respiration using high resolution O2K-FluoRespirometer, mito morphology using electron microscopy, and biochemical assays. Cardiomyocytes and H9c2 cells were used for in vitro mechanistic studies. Results: Compared to controls, PH rats show increased RV systolic pressure and RV dysfunction. Mito complex I-coupled oxidative phosphorylation and electron transfer capacity are significantly reduced in RV of PH rats (O 2 flux per mass: 216±21 vs. 90±19 and 209±22 vs. 91±22 pmol·s-1·mg-1, respectively, p <0.01, n=6 each), which is associated with decreased ATP but no changes in mito membrane integrity, mito DNA content, or mito size. Mito c-Src kinase activity and expression are significantly reduced in RV of PH rats and in dysfunctional RV of human patients. Chemical or molecular inhibition of mito c-Src results in a similar reduction of mito respiration in culture as found in RV of PH rats and is associated with decreased ATP production. Mito c-Src phosphorylates NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2), a critical protein for complex I, and P-Tyr of NDUFV2 is reduced in RV of PH rats. Conclusions: Impaired mito bioenergetics is associated with progression of RV dysfunction. Basal mito c-Src kinase activity is required for mito bioenergetics and mito c-Src-dependent P-Tyr of NDUFV2 may serve as a regulatory mechanism in impaired RV mito bioenergetics in settings of PH.

ELP3 Acetyltransferase is phosphorylated and regulated by the oncogenic anaplastic lymphoma kinase (ALK).

Protein lysine acetylation is one of the major posttranslational modifications (PTMs) with several thousands of proteins identified to be acetylated in mammalian tissues. Mechanistic studies have revealed important functions of acetylation in the regulation of protein function. Much less is known on how the acetyltransferases themselves are regulated. In the current study, we discover that the Elongator protein 3 (ELP3) acetyltransferase is modified by tyrosine phosphorylation. We demonstrate that the anaplastic lymphoma kinase (ALK) is the major tyrosine kinase responsible for ELP3 tyrosine phosphorylation. ELP3 is phosphorylated in tumor cells expressing oncogenic NPM-ALK fusion protein. We further identify Tyr202 as the major ALK phosphorylation site in ELP3. Importantly, the introduction of Y202 phosphorylation mutant ELP3 into ALK-positive tumor cells reduced cell growth and impaired gene expression. Collectively, our study reveals a novel regulatory mechanism for ELP3, provides an example that acetyltransferase itself can be regulated by PTM, and suggests a potential target for ALK-positive cancer therapies.

Comparison of the effects of the three major tyrosine kinase inhibitors as first-line therapy for non-small-cell lung cancer harboring epidermal growth factor receptor mutations

IntroductionPatients with advanced lung adenocarcinoma harboring epidermal growth factor receptor (EGFR)-activating mutations have good response rate and longer progression-free survival (PFS) when treated with the tyrosine kinase inhibitors (TKI) compared with platinum-based chemotherapy. However, studies comparing the effectiveness of these drugs as first-line therapy in such patients are limited.ResultsWe analyzed 422 patients with EGFR-mutated advanced lung adenocarcinoma receiving first-line gefitinib (n = 195, 46.2%), erlotinib (n = 123, 29.1%), or afatinib (n = 104, 24.6%). The PFS of the afatinib group was longer (12.2 months) than that of the gefitinib group (9.8 months) (p = 0.035) but similar to that of the erlotinib group (11.4 months) (p = 0.38). In patients without brain metastasis (BM), subgroup analysis showed that the afatinib group had significantly longer PFS (13.1 months) than erlotinib (11.7 months) and gefitinib (9.8 months) groups (p = 0.010). Patients with exon 19 deletions in the afatinib and erlotinib groups had potentially long PFS (p = 0.073). Efficacy of afatinib was similar between the 30 mg and 40 mg arms (median PFS 16.1 months vs. 10.3 months; p = 0.923).ConclusionsAfatinib may be the optimal EGFR-TKI for advanced lung adenocarcinoma harboring EGFR-activating mutations, particularly in the absence of BM. Patients with exon 19 deletions taking afatinib had potentially long PFS. An afatinib dose of 30 and 40 mg has similar effect.MethodsWe conducted this retrospective study at a single medical center from January 2013 to March 2017 and used PFS to evaluate the effectiveness of gefitinib, erlotinib, and afatinib in patients with advanced lung adenocarcinoma harboring EGFR mutations.

SH3 Domain of C-Src Regulates its Dynamic Behavior in the Cell Membrane

Src family kinases are major non-receptor tyrosine kinase in cells and Src-mediated signal transduction involves various cellular functions. In activation process, Src molecules translocate to cell membrane and the subdomains in Src, SH2 and SH3 domains, are exposed. For Src to serve as a kinase, the interaction with its substrates via SH2 and/or SH3 domains is required. Although the activation mechanism of Src has been well-studied, the dynamics of Src at the cell membrane is still unclear. In this study, we examined the role of Src subdomains, especially SH2 and SH3 domains, on the dynamics of Src at the cell membrane. To achieve this, we constructed PAmCherry-tagged wild-type Src (SrcWT), SrcW121A and SrcR178A mutants that decrease the binding of Src to its substrate(s) via SH3 and SH2 domains, respectively, and traced individual Src molecules in the cell membrane with gentle activation of PAmCherry. SrcWT dynamically moved on the cell membrane in the range of 0.27±0.01 μm2/s within a few seconds. The dynamics of SrcR178A mutant was comparable with that of SrcWT, whereas SrcW121A mutant exhibited less mobility (0.16±0.01 μm2/s) at the cell membrane compared with SrcWT. Since both SrcW121A and SrcR178A mutants showed higher phosphorylation level than SrcWT, the result indicates that the less mobility of SrcW121A in the cell membrane seems not to depend upon Src activation status. We further demonstrate that SrcW121A mutant showed ∼30% increase in the Src molecules residence time at focal adhesion compared with SrcWT, which is mediated by slower dissociation from adhesion site. Taken together with enhanced localization of SrcW121A at focal adhesion, our findings show that the SH3 domain of Src molecules governs dynamics of Src at the cell membrane, which may be involved in the rapid signal transduction in cells.

Transmembrane Domain Targeting Peptide Antagonizing ErbB2/Neu Inhibits Breast Tumor Growth and Metastasis

Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.

MLS-2384, a new 6-bromoindirubin derivative with dual JAK/Src kinase inhibitory activity, suppresses growth of diverse cancer cells

Janus kinase (JAK) and Src kinase are the two major tyrosine kinase families upstream of signal transducer and activator of transcription (STAT). Among the seven STAT family proteins, STAT3 is constitutively activated in many diverse cancers. Upon activation, JAK and Src kinases phosphorylate STAT3, and thereby promote cell growth and survival. MLS-2384 is a novel 6-bromoindirubin derivative with a bromo-group at the 6-position on one indole ring and a hydrophilic group at the 3′-position on the other indole ring. In this study, we investigated the kinase inhibitory activity and anticancer activity of MLS-2384. Our data from in vitro kinase assays, cell viability analyses, western blotting analyses, and animal model studies, demonstrate that MLS-2384 is a dual JAK/Src kinase inhibitor, and suppresses growth of various human cancer cells, such as prostate, breast, skin, ovarian, lung, and liver. Consistent with the inactivation of JAK and Src kinases, phosphorylation of STAT3 was inhibited in a dose-dependent manner in the cancer cells treated with MLS-2384. STAT3 downstream proteins involved in cell proliferation and survival, such as c-Myc and Mcl-1, are downregulated by MLS-2384 in prostate cancer cells, whereas survivin is downregulated in A2058 cells. In these two cancer cell lines, PARP is cleaved, indicating that MLS-2384 induces apoptosis in human melanoma and prostate cancer cells. Importantly, MLS-2384 suppresses tumor growth with low toxicity in a mouse xenograft model of human melanoma. Taken together, MLS-2384 demonstrates dual JAK/Src inhibitory activity and suppresses tumor cell growth both in vitro and in vivo. Our findings support further development of MLS-2384 as a potential small-molecule therapeutic agent that targets JAK, Src, and STAT3 signaling in multiple human cancer cells.

Abstract B119: Mechanisms of drug resistance to molecular target therapeutics against tyrosine kinases as EGFR, c-Met, and PDGFR in GBM.

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Despite technological advances in surgery, combined with regimens of radiotherapy and new generation chemotherapy, the median survival is still 15 months. Several molecular therapeutics targeting protein tyrosine kinases have recently been attempted; however, the emerging drug resistance is a crucial issue. Here, we established cell lines acquired drug resistance to individual inhibitor for major tyrosine kinase as EGFR, c-Met, or PDGFR that highly expressed in GBM, and characterized the underlying mechanisms. While initial 50 days starting inhibitor treatment, the cells exhibited distinct morphological changes of epithelial-mesenchymal transition (EMT) concomitant with up-regulation of Snail, Slug, and Mmp-2. Of note, further continuous drug treatment produced stemness-like properties in the cells. PCR array and microarray analysis have identified marked enhancement of several stemness-related genes including Sox2 and CXCL12. These findings show that suppression of EMT and/or stemness features of GBM may be an effective strategy to avoid the acquisition of resistance for molecular target therapy in GBM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B119. Citation Format: Masumi Tsuda, Lei Wang, Mishie Tanino, Taichi Kimura, Hiroshi Nishihara, Shinya Tanaka. Mechanisms of drug resistance to molecular target therapeutics against tyrosine kinases as EGFR, c-Met, and PDGFR in GBM. [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 B119.

M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

Activated Mutant NRasQ61K Drives Aberrant Melanocyte Signaling, Survival, and Invasiveness via a Rac1-Dependent Mechanism

Around a fifth of melanomas exhibit an activating mutation in the oncogene NRas that confers constitutive signaling to proliferation and promotes tumor initiation. NRas signals downstream of the major melanocyte tyrosine kinase receptor c-kit and activated NRas results in increased signaling via the extracellular signal–regulated kinase (ERK)/MAPK/ERK kinase/mitogen-activated protein kinase (MAPK) pathways to enhance proliferation. The Ras oncogene also activates signaling via the related Rho GTPase Rac1, which can mediate growth, survival, and motility signaling. We tested the effects of activated NRasQ61K on the proliferation, motility, and invasiveness of melanoblasts and melanocytes in the developing mouse and ex vivo explant culture as well as in a melanoma transplant model. We find an important role for Rac1 downstream of NRasQ61K in mediating dermal melanocyte survival in vivo in mouse, but surprisingly NRasQ61K does not appear to affect melanoblast motility or proliferation during mouse embryogenesis. We also show that genetic deletion or pharmacological inhibition of Rac1 in NRasQ61K induced melanoma suppresses tumor growth, lymph node spread, and tumor cell invasiveness, suggesting a potential value for Rac1 as a therapeutic target for activated NRas-driven tumor growth and invasiveness.

A New HPLC-UV Validated Method for Therapeutic Drug Monitoring of Tyrosine Kinase Inhibitors in Leukemic Patients

Development and validation of simple, rapid, and reliable high-performance liquid chromatography (HPLC)-UV method for quantification of major tyrosine kinase inhibitors, imatinib, dasatinib, and nilotinib, in human plasma is presented. Chromatographic separation of the drugs is achieved on an RP-C(18) column at flow rate of 0.9 mL/min at 35°C; eluate is monitored at 267 nm. Mean intra-day and inter-day precision for all compounds are 2.5 and 13.3%; mean accuracy is 13.9%; extraction recovery ranges within 40.24 and 81.81%. Calibration curves range from 10 to 0.005 μg/mL. Limits of detection are 10 ng/mL for imatinib and nilotinib, 50 ng/mL for dasatinib; limits of quantitation are 50 ng/mL for imatinib and nilotinib, 100 ng/mL for dasatinib. Although this method allows the detection of dasatinib, levels found in patients plasma are close to the limit of detection, then below the limit of quantitation. Quantification with HPLC-mass spectrometry, then, is required for dasatinib to give a correct evaluation. In conclusion, the sensitivity of this new method is sufficient to perform therapeutic monitoring and pharmacokinetic studies of imatinib and nilotinib but not dasatinib in CML patients.

Carcinoma epidermóide do pulmão: Polissomia e amplificação do cromossoma 7 e do gene EGRF com forma wild type nos exões 19 e 21

PurposeThe epidermal growth factor receptor (EGFR) is overexpressed in the majority of nonsmall- cell lung cancers (NSCLC) and is a major target specific EGFR tyrosine kinase inhibitors (TKIs) developed and used for the treatment of advanced NSCLC. A number of biological factors are also associated with EGFR-TKIs responsiveness. This study was focused on EGFR somatic mutations and amplifications in squamous cell lung cancer. Material and methodsRepresentative sections of squamous cell carcinoma were selected from 54 surgical specimens from formalin-fixed paraffin-embedded tissues and submitted to TMA construction. Determination of EGFR protein expression was done by immunohistochemistry(IHC) (Zymed, Laboratories). Fluorescence in situ hybridization (FISH) was performed with LSI EGFR/CEP 7 (Vysis; Abbott Molecular, USA). Genomic DNA was extracted from 48 cases and exon 19 was amplified by polymerase chain reaction (PCR) for search deletions and point mutations for exon 21. All cases expressed high weigh cytokeratin and were observed negativity for CK7, CD56 and chromogranin. ResultsEGFR protein overexpression was identified in 49 cases, by the application of Hirsh's scoring system. The chromosome 7 and EGFR gene were analyzed by FISH and scored according to Cappuzzo's method that showed high polysomy in 31 cases and amplification in 7 cases. Deletion in exon 19 of EGFR was detected in 3 cases of 48 samples; the exon 21 of EGFR was expressed in its wild type by RFLP in all cases. ConclusionsDetection of common EGFR deletion and mutation showed to be a rare event in Squamous cell carcinoma of the lung. While EGFR mutation is the most effective molecular predictor or sensitivity in patients with advanced NSCLC submitted to EGFR-TKIs treatment, amplification and polysomy is the most effective molecular predictor for EGFR-TKIs responsiveness in squamous cell carcinoma, when valida ted isolated from the group of NSCLC.

Carcinoma epidermóide do pulmão: Polissomia e amplificação do cromossoma 7 e do gene EGRF com forma wild type nos exões 19 e 21

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in the majority of nonsmall-cell lung cancers (NSCLC) and is a major target specific EGFR tyrosine kinase inhibitors (TKIs) developed and used for the treatment of advanced NSCLC. A number of biological factors are also associated with EGFR-TKIs responsiveness. This study was focused on EGFR somatic mutations and amplifications in squamous cell lung cancer. Material and methods: Representative sections of squamous cell carcinoma were selected from 54 surgical specimens from formalin-fixed paraffin-embedded tissues and submitted to TMA construction. Determination of EGFR protein expression was done by immuno-histochemistry(IHC) (Zymed, Laboratories). Fluorescence in situ hybridization (FISH) was performed with LSI EGFR/CEP 7 (Vysis; Abbott Molecular, USA). Genomic DNA was extracted from 48 cases and exon 19 was amplified by polymerase chain reaction (PCR) for search deletions and point mutations for exon 21. All cases expressed high weigh cytokeratin and were observed negativity for CK7, CD56 and chromogranin. Results: EGFR protein overexpression was identified in 49 cases, by the application of Hirsh’s scoring system. The chromosome 7 and EGFR gene were analyzed by FISH and scored according to Cappuzzo’s method that showed high polysomy in 31 cases and amplification in 7 cases. Deletion in exon 19 of EGFR was detected in 3 cases of 48 samples; the exon 21 of EGFR was expressed in its wild type by RFLP in all cases. Conclusions: Detection of common EGFR deletion and mutation showed to be a rare event in Squamous cell carcinoma of the lung. While EGFR mutation is the most effective molecular predictor or sensitivity in patients with advanced NSCLC submitted to EGFR-TKIs treatment, amplification and polysomy is the most effective molecular predictor for EGFR-TKIs responsiveness in squamous cell carcinoma, when validated isolated from the group of NSCLC.Rev Port Pneumol 2010; XVI (3): 453-462

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients’ follow-up. We have developed a liquid chromatography–tandem mass spectrometry method (LC–MS/MS) requiring 100μL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1–104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3–9.4%), accurate (−9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10ng/mL). This is the first broad-range LC–MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Role of Focal Adhesion Kinase Ser-732 Phosphorylation in Centrosome Function during Mitosis

Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis.

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by α- and γ-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.

Non-neuronal cholinergic system and signal transduction pathways mediated by band 3 in red blood cells

Non-neuronal acetylcholine (ACh) and acetylcholinesterase (AChE) have been recognized in the past. Vascular ACh has been associated by us with the regulation of microcirculatory flow by modulating nitric oxide (NO) intracellular mobilization, metabolism (NOx) and release from erythrocytes, as well as the glycolytic flux. Velnacrine maleate is a well-known AChE inhibitor which plays a competitive role by decreasing NO-mediated erythrocyte responses. A plausible hypothesis to explain the mechanisms underlying those events hinges on the NO translocation among nitrosylated molecules and phosphorylated/dephosphorylated states of band 3 protein, processed by major tyrosine-kinases (PTK: p72syk, p53/56lyn and p59/61hck) and phosphotyrosine-phosphatases (PTP). To assess this hypothesis under the influence of AChE effectors (acetylcholine/velnacrine), blood samples from healthy donors were harvested and Western blot analysis was subsequently used to determine the degree of band 3 phosphorylation, in the presence and absence of PTK/PTP inhibitors. NO and nitrites/nitrates were quantified using an amperometric method and the Griess Reaction, respectively, in erythrocyte suspensions. Measurements of erythrocyte metabolites (2,3-bisphosphoglycerate; glyceraldehyde 3-phosphate dehydrogenase; glucose-6-phosphodehydrogenase; lactate), hemoglobin and cyclic nucleotides were conducted afterwards. Increased levels of phosphorylated-band 3 obtained upon p72syk inhibition suggest p59/61hck and p53/56lyn as secondary involved kinases. As to NO/NOx quantification, in the presence of PTKi we reported higher levels with velnacrine-AChE, as opposed to acetylcholine-AChE. Calpeptin, a PTP inhibitor which triggers full band 3-phosphorylation, led to the opposite NO mobilization, being reinforced by ACh. Oxy-hemoglobin, glyceraldehyde 3-phosphate dehydrogenase and glucose-6-phosphodehydrogenase were found to decrease with ACh, whereas P50, lactate and both cGMP/cAMP happened to increase. Changes on human erythrocyte NOx mobilization and metabolic fluxes occur under influence of non-neuronal ACh/AChE, in turn dependent on the degree of band 3-phosphorylation. Since these vascular events may potentially change under pathological conditions, coadjuvant drugs could become accessible in the setting of microcirculation disease.

Modeling targeted inhibition of wild type and mutated c-MET receptor

14010 Background: c-MET belongs to the semaphorin superfamily of signaling proteins, containing three protein families (semaphorins, plexins, c-MET and RON) that have central roles in cell signaling. The c-MET receptor tyrosine kinase is involved in regulating cell growth/proliferation, survival, angiogenesis, cell scattering, cell motility and migration. Mutations in c-MET have been identified in various human cancers including lung cancer and papillary renal cell carcinomas. c-MET mutations occur within the extracellular seven-blade β-propeller fold sema domain (E168D, L229F, S325G, N375S), juxtamembrane domain (R988C, T1010I), and kinase domain (M1268T). We hypothesized that these mutations would have differential effects on the kinase inhibition. Methods: We modeled the various c-MET mutations from different functional domains of the receptor using a G418-resistant stable Cos-7 transfection cell system to determine their effect on sensitivity to a selective c-MET kinase inhibitor SU11274. Sensitivity to SU11274 inhibition was assayed by phospho-immunoblotting using phospho- specific antibody against the major tyrosine kinase phosphorylation epitopes pY1234/1235 of the c-MET kinase in vitro. Results: First, we identified that mutations in the sema and juxtamembrane domain were activating as defined by ligand-independent constitutive receptor activation. SU11274 was capable of inhibiting ligand induced signaling through the wild-type c-MET as well as mutant c-MET receptors harboring mutations in the sema, juxtamembrane and tyrosine kinase domain. However, SU11274 inhibition of mutant c-MET was mutation-dependent, with the juxtamembrane domain mutations R988C and T1010I resulting in a receptor form that was less sensitive to SU11274. Mutations in the sema and kinase domain also resulted in varying sensitivity to inhibition by SU11274 inhibition. Conclusions: Mutations in the sema and juxtamembrane domain of c-MET result in receptor activation. The small molecule inhibitor SU11274 is active against wild type and mutated c- MET receptor. Further studies to characterize the signaling effects and the mechanism of sensitivity and resistance of c-MET mutations to specific inhibitors are crucial in the successful development of therapeutic c-MET and mutant c-MET inhibitors. No significant financial relationships to disclose.

Tyrosine kinase receptors are crucial for normal β-cell development and function

Signaling pathways play critical roles in most physiological and pathological processes and convert an extracellular stimulus into a change of function in the recipient cell. Intracellular messages originate from the activation of membrane receptors by a variety of ligands, such as hormones, nutrients or growth factors. The receptors subsequently interact with specific intracellular cascades, triggering the phosphorylation of cell effectors. In the pancreas, these processes control the organogenesis, maintenance and function of endocrine cells within the islets. Growth factors acting through tyrosine kinase receptors play a prominent role among the multitude of signaling pathways active in pancreatic β cells. Deregulation of these processes leads to the development of disorders such as hypoglycemia or diabetes. This review will describe recent advances made on the understanding of the roles of major tyrosine kinase receptors in pancreatic β-cell physiology.

DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.

Src tyrosine kinase-dependent migratory effects of antithrombin in leukocytes

Tyrosine kinases are known to play a critical role in the regulation of leukocyte function. Antithrombin mediates its effects via syndecan-4 which is known to be linked to the Src tyrosine kinases. In this study, we investigated the role of Src tyrosine kinases in antithrombin-regulated leukocyte migration and Src tyrosine kinase phosphorylation in response to stimulation with antithrombin. Neutrophils and monocytes obtained from forearm venous blood were pre-treated by various Src-family selective tyrosine kinase inhibitors with or without antithrombin followed by washing and assessment of their migratory response toward antithrombin, interleukin-8, or RANTES using Boyden microchemotaxis chambers. Activation status of the two major Src tyrosine kinase phosphorylation sides Tyr416 and Tyr527 was tested using Western blot analysis. Dose-dependent reversal of the antithrombin-mediated effects on neutrophil and monocyte migration was induced by the selective Src kinase inhibitors PP1 and PP2. In Western blot analyses, antithrombin increased Tyr416 and decreased Tyr527 phosphorylation of Src tyrosine kinases in a time- and dose-dependent manner. Moreover, co-incubation with antithrombin lowered the level of RANTES-induced Tyr416 phosphorylation. Therefore, Src tyrosine kinases linked to signaling of antithrombin-binding sites on leukocytes may play an important role in modulating effects on cells function.