Receptor Tyrosine Kinase Domain Research Articles

Structural Aspects of Transmembrane Domain Interactions of Receptor Tyrosine Kinases

Specific helix-helix interactions of single-span transmembrane domains of receptor tyrosine kinases are critical for their lateral dimerization and biological function. Establishing structure-function relationship as well as rational drug design requires precise structural information about this class of biologically significant bitopic membrane proteins. A combination of NMR, optical spectroscopy, protein engineering and molecular modelling made it possible studies of the nature and the mechanisms of important transmembrane helix-helix interactions inside the supramolecular complexes mimicking membrane environment. Using a robust strategy we investigated recombinant transmembrane fragments from different families of receptor tyrosine kinases: EphA, ErbB and FGFR, which play important roles in normal and in pathological conditions of human organism by providing cell signalling, maintaining cellular homeostasis and controlling cell fate. We established spatial structure and internal dynamics of the homo- and heterodimeric transmembrane domains, characterized diverse transmembrane helix-helix packing interfaces and obtained detailed picture of intra- and intermolecular interactions in membrane. The already available information about structural-dynamic properties of the dimeric transmembrane domains of studied receptor tyrosine kinases along with the available biophysical and biochemical data provides useful insights into their functioning in the human organism at the atomic level.

Effect of FGFR3 Juxtamembrane Domain on FGFR3 Dimerization

Receptor Tyrosine Kinases have four distinct domains: extracellular (EC), transmembrane (TM), juxtamembrane (JM), and catalytic (CAT). Receptor Tyrosine Kinase (RTK) dimerization is critical for RTK function, and disregulation of ligand-independent dimerization of RTKs is known to be the underlying cause for a number of human pathologies. Yet, the exact mechanism of RTK dimerization, and the roles of the four RTK domains in the dimerization process are unknown. To investigate the role of the juxtamembrane domain in ligand-independent homodimerization of RTKs, we are comparing the dimerization of two truncated FGFR3 constructs which both lack the catalytic domain, EC+TM+JM and EC+TM. We assess dimerization in single membrane-derived vesicles using the quantitative imaging FRET (QI-FRET) method [Li et al., 2008, Chen et al., 2010]. The results show that the juxtamembrane domain of FGFR3 increases the measured FRET efficiency and hence contributes to the energetics of lateral dimerization of the FGFR3 receptor. Li E,Placone J,Merzlyakov M, Hristova K (2008) Quantitative measurements of protein interactions in a crowded cellular environment.Anal Chem 80:5976-5985. Chen L, Novicky L, Merzlyakov M, Hristov T, Hristova K (2010) Measuring the energetics of membrane protein dimerization in mammalian membranes. J Am Chem Soc 17;132(10):3628-35.

Role of EGFR Juxtamembrane Domain in EGFR Activation

The intracellular juxtamembrane domain of several receptor tyrosine kinases (RTKs) has been proposed to play an autoinhibitory role. The epidermal growth factor receptor (EGFR) has been widely used as a model in studies of the RTK activation mechanism. Extensive efforts have been devoted to studying the functions of EGFR's different domains; however, the role of the juxtamembrane domain in EGFR phosphorylation and activation is yet to be clarified. In this work, we replaced the 30 amino acids (674-704) of the EGF juxtamembrane domain with ten repeats of Glycine-Glycine-Serine (GGS). Preliminary results from western blots showed decreased phosphorylation of Tyr1148 in the EGFR-GGS chimeric receptor, which suggests that the juxtamembrane domain of EGFR plays an activating role rather than an inhibitory one. Currently we are using ligand titration and Foster resonance energy transfer (FRET) experiments to further explore the difference between wild type EGFR and the EGFR-GGS chimeric receptor. Results from this work will yield further insights into the function of the juxtamembrane domain in the EGFR activation process.

An Engineered Cell Surface Polypeptide for Selection, Tracking and Ablation of Genetically Modified Hematopoietically Derived Cells Based on Human EGFR.

AbstractAbstract 1469Hematopoietic cell-based therapies, including genetically manipulated cell products derived from either hematopoietic stem cells or T cells, is an emerging area in applied biotechnology. In both of these venues, a variety of genetic engineering approaches are being studied to endow cells with novel attributes, to increase their therapeutic potency and/or safety. Common to the field of ex vivo cellular genetic engineering is the need to purify cells that express desired quantities of therapeutic transgene(s) and cull out non-expressing cells that either lack transgene endowed therapeutic activity or safety features. However, current drug selection strategies are associated with prolonged ex vivo culture that drives terminal differentiation of the T cells, which has in turn been found to be associated with impaired antitumor efficacy of adoptively transferred CD8+ T cells in vivo. Thus, we were interested in developing a single transgene encoded polypeptide that can serve both as an ex vivo selection epitope and in vivo tracking marker/target for mAb-mediated cell ablation, while fulfilling the criteria of being functionally inert, non immunogenic, and amenable to commercially available cGMP-grade selection systems appropriate for clinical use. Here we describe a truncated human EGFR polypeptide (huEGFRt) devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase domains. Retained features of huEGFRt include type I transmembrane cell surface localization and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR mAb, cetuximab/Erbitux™. [Display omitted] Applying this system to cellular immunotherapy, we designed lentiviral vector prototypes housing multifunctional constructs combining huEGFRt with CD19-specific chimeric antigen receptors (CARs), and demonstrate that biotinylated-cetuximab immunomagnetic selection of transduced human T cells results in coordinate enrichment of CAR+ cells from 2% to over 90%. The huEGFRt-mediated selection did not affect the phenotype (i.e., TCR, CD3, CD4, CD8, CD28, and granzyme A expression), the in vitro expansion potential, nor the in vivo engraftment fitness (upon transfer into immunodeficient mice) of the T cells. Direct examination of EGF-binding and phospho-tyrosine analysis confirmed that this selection marker is functionally inert and has no negative effect on the T cell product. In addition, cytotoxicity against B cell malignancies and IFN-g/TNF-a production through the CD19-specific CAR was dramatically enhanced in the huEGFRt-selected population. The utility of huEGFRt in tracking the gene modified, transferred cells in vivo within easily obtained human tissues such as blood, bone marrow and tissue biopsies was then also proven via detection of huEGFRt using multiparameter flow cytometric analysis or FDA approved immunohistochemical techniques/reagents. In addition, we were able to demonstrate that Erbitux™ could mediate ADCC of huEGFRt+ T cells in vitro and inhibit the growth of huEGFRt+ CTLL2 cells in NOD/Scid mice, supporting the use of huEGFRt as a suicide gene via cetuximab-mediated ADCC after adoptive transfer. Together these data suggest that huEGFRt is a superior selection marker for any transduction system that can be applied to the generation of cell products for hematopoietic cell-based medical therapies. Disclosures:No relevant conflicts of interest to declare.

110 Targeting focal adhesion kinase (FAK) in primary human schwannoma

Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory “molecular brake” mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

Clinical impact of amphiregulin expression in patients with epidermal growth factor receptor (EGFR) wild‐type nonsmall cell lung cancer treated with EGFR‐tyrosine kinase inhibitors

In patients with nonsmall cell lung cancer (NSCLC), several studies have demonstrated a positive correlation between somatic mutation in the epidermal growth factor receptor (EGFR) tyrosine kinase domain and clinical outcomes with the use of EGFR tyrosine kinase inhibitors (TKIs). However, some patients with wild-type (WT) EGFR also responded to EGFR TKIs and remained stable. Recently, amphiregulin (AR) has been suggested as a predictive marker for EGFR TKIs in patients with WT EGFR-positive NSCLC. The objective of the current study was to evaluate the association between AR expression and the efficacy of using EGFR TKIs in the treatment of patients with WT EGFR-positive NSCLC. Seventy-three patients with WT EGFR-positive NSCLC received treatment with gefitinib or erlotinib between May 2005 and December 2008. AR expression was assessed by immunohistochemistry. The clinical response to EGFR TKIs was reassessed for all patients as follows: 16 of 73 patients had a partial response (21.9%), 12 patients had stable disease (16.5%), and 45 patients had progressive disease (61.6%). AR expression was positive in 24 of 40 patients (60%). The ability to achieve disease control did not differ significantly between AR-positive patients and AR-negative patients (P = .188). At a median follow-up of 25.4 months (range, 10.5-53.3 months), progression-free survival was 8.1 weeks in AR-positive patients and 4 weeks in AR-negative patients (P = .025), and overall survival was significantly longer in AR-positive patients than in AR-negative patients (12.2 months vs 4.1 months; P = .001). The current results suggested that patients with WT EGFR-positive NSCLC who have AR-positive tumors may benefit clinically from treatment with EGFR TKIs, indicating that AR expression may be a potential marker for the selection of EGFR-TKI treatment for patients with WT EGFR-positive NSCLC.

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if the inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.

The sphingolipid‐rich rafts of ALK+ lymphomas downregulate the Lyn‐Cbp/PAG signalosome

Human anaplastic lymphoma kinase (ALK) + lymphomas express the constitutively active ALK as a fusion protein that drives several survival pathways. The catalytic domain of the anaplastic receptor tyrosine kinase is frequently fused with the nuclear localization protein nucleophosmin but may also fuse with other proteins that associate it with other subcellular structures. Similarly to other B human lymphomas, ALK+ lymphomas express the Cbp/PAG adaptor protein and the non-receptor Lyn kinase in the plasma membrane. In the majority of human lymphomas, the Cbp/PAG adaptor and the Lyn kinase constitute an oncogenic signalosome that serves as a membrane anchor for other signaling enzymes and transcription factors. We show that ALK+ lymphoma membranes harbor sphingolipid-rich microdomains (rafts) in which Lyn is poorly active. However, Lyn activity and consequently Cbp/PAG tyrosine phosphorylation can be restored by extracting sphingolipids from ALK+ lymphoma plasma membranes. In the membrane environment of ALK+ lymphoma rafts, where the glycosphingolipid to signaling protein ratio is higher than in B-NHL rafts, the Lyn activity is suboptimal and does not allow the formation of an efficient Lyn-Cbp/PAG signalosome.

Abstract 1190: Differences in responsiveness of drug-sensitive and drug-resistant human bladder cancer cells to specific EGFR inhibitors

Abstract Bladder cancer is a common genitourinary malignant disease in the USA. EGFR and/or HER2 are overexpressed in these cancers, and EGFR/HER2 inhibitors are suggested as treatment. Gefitinib (i.e., Iressa) is a selective inhibitor of the epidermal growth factor receptor (EGFR) and Lapatinib is a dual inhibitor of both the EGFR and HER2/neu (Human EGFR type 2) receptor. Both compounds compete with the binding of ATP to the tyrosine kinase domain of the respective receptors to inhibit autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a problem. The purpose of this study was to compare the signaling pathway(s) induced by Gefitinib and Lapatinib, in UM-UC-5 (Gefitinib-sensitive) and UM-UC-14 (Gefitinib-resistant) bladder cancer cells and to identify molecular markers that might be useful predictors of efficacy. Each cell line was cultured to 90% confluence and then incubated 24 h in serum-free media. They were treated, respectively, with 2.5 μM Gefitinib or Lapatinib for 24 h and harvested. Cell lysates were subjected to Proteome Profiler™ Arrays (R&D, Minneapolis, MN) including RTK, phospho-MAP kinase, phospho-kinase, and apoptosis arrays. Results indicate that Gefitinib and Lapatinib had differential effects on proteins known to play a role in apoptosis. Iressa appeared to decrease anti-apoptotic Bcl2, whereas Lapatinib increased pro-apoptotic Bax expression. Lapatinib, but not Iressa, also suppressed anti-apoptotic clusterin. Both agents decreased anti-apoptotic claspin, survivin, and XIAP expression but the effect of Lapatinib was 2- to 8-fold greater than Iressa. Notably, in the drug-resistant UM-UC-14 cells, Iressa had little affect whereas Lapatinib induced pro-apoptotic BAD and inhibited expression of anti-apoptotic Bcl2, Bcl-XL, and clusterin. Perhaps the most notable difference between these two drugs, was the effectiveness of Lapatinib in suppressing EGFR/HER2 expression in both cell lines. In the sensitive line, Lapatinib suppressed EGFR/HER2 expression 2-3 fold more than did Iressa and was almost as effective in the resistant line, whereas Iressa had no effect on UM-UC-14 cells. In contrast, Iressa was at least 8-fold more effective than Lapatinib in suppressing ERK1/2 expression in drug-sensitive cell lines but neither compound affected ERKs in resistant cells. Overall, the effect of these compounds on MAP kinase signaling appeared to be similar in sensitive cells; however, Lapatinib was able to suppress some MAP kinase signaling in the resistant line. Other notable differences included increased phosphorylation of p53, p27 and p70S6 kinase only by Lapatinib and decreased phosphorylation of eNOS only by Iressa in sensitive cells. Overall results suggest that human bladder cancer cells display some similarity but also marked heterogeneity in response to “specific” EGFR inhibitors ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1190.

SpTie1/ 2 is expressed in coelomocytes, axial organ and embryos of the sea urchin Strongylocentrotus purpuratus, and is an orthologue of vertebrate Tie1 and Tie2

A full length cDNA sequence expressed in coelomocytes shows significant sequence match to vertebrate Tie1 and Tie2/TEK. Vertebrate Tie2/TEK is the receptor for the angiopoietins and plays an important role in angiogenesis and hematopoiesis, whereas Tie1 regulates the activity of Tie2. The deduced sequence of the SpTie1/2 protein has a similar order and organization of domains to the homologous vertebrate proteins including a highly conserved receptor tyrosine kinase domain in the cytoplasmic tail. The N terminus of the ectodomain has one immunoglobulin (Ig)-Tie2_1 domain, followed by an Ig domain, four epidermal growth factor domains, a second Ig domain, and three fibronectin type III domains. The SpTie1/ 2 gene is expressed in coelomocytes and the axial organ, whereas other organs do not show significant expression. The timing of embryonic expression corresponds with the differentiation of blastocoelar cells, the embryonic and larval immune cells. Searches of the sea urchin genome show several gene models encoding putative ligands and signaling proteins that might interact with SpTie1/2. We speculate that SpTie1/2 may be involved in the proliferation of sea urchin immune cells in both adults and embryos.

Structural Stability and Binding Efficiency of Strong and Weak Interactions in Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Domain of Human and Drosophila

Epidermal growth factor receptor (EGFR) is a cell membrane receptor serving as a molecular target for Non Small Cell Lung cancer (NSCLC). The aim of the study was to analyze the weak ion interactions taking place in the tyrosine kinase domain of Human and Drosophila EGFR. This study throws light on the environment preferability, stabilizing residues taking part in weak and strong interactions of EGFR TK domain. Analysis of short, medium and long range contacts showed that the cation-� interactions are mainly formed by long-range contacts, whereas CH…OC interactions are formed by both long and short range interactions. Except Pro, other non-polar hydrophobic amino acids in Drosophila EGFR TK domain were present in buried environment. However, in humans other than Pro and Ala, which were involved in SS (CH…OC) and SM (NH…OC) interactions, other non-polar hydrophobic amino acid preferred to be in buried environment. The results observed in this study will be useful for understanding the contribution of weak and strong interactions to the stability of EGFR TK domain. Further, the weak interactions have distinct roles in the stability of EGFR TK domain in addition to other conventional strong interactions. The results can lead to the identification of novel targets for NSCLC drugs which in turn will serve as a major break through in the treatment of NSCLC. methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine), both orally available, low molecular weight, reversible and selective EGFR-TKIs, were used as advanced EGFR- targeted drugs in NSCLC treatment. They blocked signal

Structural Stability and Binding Efficiency of Strong and Weak Interactions in Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Domain of Human and Drosophila

Structural Stability and Binding Efficiency of Strong and Weak Interactions in Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Domain of Human and Drosophila

9154 Clinical outcomes in patients with EGFR mutations: pooled analysis of NSCLC patients treated with either an EGFR TKI or chemotherapy

Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory “molecular brake” mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

Three cases of severe hepatic impairment caused by erlotinib

Lung cancer is the second-most common cancer in men and women, accounting for about 13% of all diagnosed cancers. It is, however, the leading cause of cancer-related deaths in industrialized countries, exceeding the mortality from breast, colorectal, and prostate cancer combined. Erlotinib is an orally administered, quinazoline-based agent that competes with adenosine triphosphate for binding to the intracellular catalytic domain of the epidermal growth factor receptor (HER1/EGFR) tyrosine kinase, inhibiting phosphorylation. Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation and its associated downstream signaling events, and it may block tumorigenesis that is mediated by inappropriate HER1/EGFR signaling [1]. In November 2004, the U.S. Food and Drug Administration (FDA) approved erlotinib as a single-agent treatment for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure with at least one standard chemotherapy regimen. Erlotinib is better tolerated and causes fewer adverse reactions than cytotoxic drugs. The most common adverse reactions in patients are rash (acneform eruptions) and diarrhoea [2]. The majority of hepatotoxicity presents as mild or moderate hepatic dysfunction; severe hepatic impairment has been documented rarely. Here, we report three cases of severe hepatic impairment which was caused by erlotinib. Erlotinib is cleared predominantly by the liver and is metabolized primarily by oxidation of CYP3A4, which is excreted in bile [3]. CYP3A4 exhibits remarkable interindividual activity as high as 20-fold. Genetic polymorphisms are a common source of these differences in the context of drug metabolism [4]. There are three mutations of CYP3A4 in the Chinese population that have been reported to attenuate CYP3A4 activity. These mutations include A13989G, C15820G and an A17776 insertion [5]. Additionally, CYP2D6 belongs to the cytochrome P450 oxidase family and is responsible for the majority of the interindividual variability in drug metabolism. There are four phenotypes of CYP2D6: poor metabolizer (PM), intermediate metabolizer (IM), extensive (normal) metabolizer (EM), and ultrarapid metabolizer (UM). If a substrate of the enzyme is given to a patient as medication, and if the patient has impaired CYP2D6, the patient will accumulate the drug and experience severe side effects. In contrast, for the UM patient, drug concentrations might be too low to have a therapeutic effect. Thus, assessing the patient's phenotype will help determine the optimal dose of a drug. One mutation in the CYP2D6 coding sequence that frequently occurs in the Chinese population is C188T(CYP2D6*10) [6].Therefore, we analyzed DNA samples from two subjects by polymerase chain reaction and direct sequencing of three mutations in the CYP3A4 coding sequence and one mutation in the CYP2D6 coding sequence. Primer design, polymerase chain reaction, and CYP3A4 and CYP2D6 sequencing were performed as described [5, 6].

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

Ephrin Receptor Expression in the Embryonic Bursa of Fabricius

Utilizing degenerate PCR primers complementary to conserved regions within the kinase domain of Receptor Tyrosine Kinase (RTK) genes, the goal was to compare the full complement of RTK expressed (mRNA level) between two developmental stages of bursal B-cells. We identified 21 different RTK cDNAs out of a total of 235 cDNAs sequenced between the two B-cell stages. The dataset revealed the expression of RTK genes important in developmental processes, namely the ephrin receptors (Eph). The ephrin receptors and the ephrin ligands play critical roles in embryo development by controlling the organization of cells in tissues and by inducing cellular differentiation.

Dimeric Quaternary Structure of the Prototypical Dual Specificity Phosphatase VH1

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 A resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced approximately 39 A away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin alpha5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-gamma signal transduction and antiviral response.

The β-Catenin Axis Integrates Multiple Signals Downstream from RET/Papillary Thyroid Carcinoma Leading to Cell Proliferation

RET/papillary thyroid carcinoma (RET/PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC stimulates the beta-catenin pathway. By stimulating PI3K/AKT and Ras/extracellular signal-regulated kinase (ERK), RET/PTC promotes glycogen synthase kinase 3beta (GSK3beta) phosphorylation, thereby reducing GSK3beta-mediated NH(2)-terminal beta-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with beta-catenin and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)beta-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3beta complex and activates the transcription factor T-cell factor/lymphoid enhancer factor. Moreover, through the ERK pathway, RET/PTC stimulates cyclic AMP-responsive element binding protein (CREB) phosphorylation and promotes the formation of a beta-catenin-CREB-CREB-binding protein/p300 transcriptional complex. Transcriptional complexes containing beta-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC-expressing cells. Silencing of beta-catenin by small interfering RNA inhibits proliferation of RET/PTC-transformed PC Cl3 thyrocytes, whereas a constitutively active form of beta-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on beta-catenin to stimulate cell proliferation.

Effect of Short Transmembrane Peptides on the Activation and Dimerization of an FGFR3 Pathogenic Mutant

Mutations in the transmembrane domains of receptor tyrosine kinases (RTKs) are implicated in many human diseases. For example, fibroblast growth factor receptor 3 (FGFR3) carrying an A391E mutation in the transmembrane (TM) region is associated with bladder cancer and Crouzon syndrome with acanthosis nigricans. Previous work has shown that the isolated FGFR3 TM domains dimerize in detergent micelles and in lipid bilayers. Therefore, we are exploring whether the TM domain of FGFR3 can inhibit the pathogenic effects of the A391E mutation. Our preliminary data show that the activation level of a chimeric Neu_FGFR3 receptor carrying the A391E mutation could be inhibited by co-expressing short mutant FGFR3 TM peptides. We also observe a decrease in dimerization propensity of the receptors due to the co-expression. Currently, we are investigating the specificity and the efficiency of the inhibition by using TM peptides from other RTKs. The TM peptides which can specifically and efficiently inhibit the activation and dimerization of FGFR3 mutants are possible candidates for future therapies for diseases linked to FGFR3 TM domain mutations.

EGFR Mutations are More Frequent in Well-Differentiated than in Poor-Differentiated Lung Adenocarcinomas

Somatic mutations in epidermal growth factor receptor (EGFR) tyrosine kinase domain, particularly deletions in exon 19 and point mutation in exon 21, are associated with clinical outcome in patients with lung adenocarcinoma, suggesting that EGFR mutation would have an important role in clinical decision making. DNA was extracted from the excised specimens of 60 lung adenocarcinoma patients with phenol-chloroform and ethanol precipitation. Exon 19 and 21 were amplified by PCR, and direct sequenced from both sense and antisense directions. EGFR somatic mutations were present in 13 of 60 patients (21.67%), including seven cases of in-frame deletion in exon 19 around codon 746 and six cases of amino acid substitution in exon 21. Exon 21 mutation is more frequent in adenocarcinomas with bronchi-alveolar component than exon 19 deletions. Mutations were more prevalent in well-differentiated adenocarcinomas (9/27, 33.33%) than in moderate to poor-differentiated adenocarcinomas (4/33, 12.12%) (P < 0.05). Adenocarcinomas with bronchi-alveolar components had higher mutation frequency (8/22,36. 36%) than those without bronchi-alveolar components (5/38, 13.16%) (P < 0.05). In this study, female patients had more mutation rate than male patients. This trend was also observed in the patients with pathologic stage I-II compared with stage III-IV, but neither of them was statistically significant. Patients with cisplatin-based adjuvant chemotherapy had no significantly prolonged survival compared with single radical resection. But patients with EGFR mutation had relative longer survival. In conclusion, our study suggest that EGFR mutations may be a valuable prognostic factor for disease free survival of surgically treated lung adenocarcinoma patients independently from adjuvant chemotherapy.