Receptor Tyrosine Kinase Domain Research Articles

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.

Laser Capture Microdissection and Protein Microarray Analysis of Human Non-small Cell Lung Cancer: Differential Epidermal Growth Factor Receptor (EGPR) Phosphorylation Events Associated with Mutated EGFR Compared with Wild Type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.

Systematic genomic screen for tyrosine kinase mutations in CLL

Systematic genomic screen for tyrosine kinase mutations in CLL

Dual-Agent Molecular Targeting of the ErbB2 Receptor: Killing One Bird With Two Stones

The phase I study by Storniolo et al in this issue of Journal of Clinical Oncology, which evaluates the combination of lapatinib (a selective small-molecule ErbB1 and ErbB2 tyrosine kinase inhibitor) and trastuzumab (an antibody blocking the extracellular domain of ErbB2) in patients with advanced ErbB2-positive breast cancer, is the first published study to our knowledge examining dual-agent molecular targeting of the ErbB2 receptor. The study demonstrated that there was clinical activity in a group of heavily pretreated patients and—using a toxicity end point—determined an optimally tolerated regimen for this combination of agents. The epidermal growth factor family of receptors plays a key role in regulating growth, survival, and resistance to chemotherapy in many epithelial malignancies. Approximately 25% of breast cancers overexpress ErbB2 or demonstrate ErbB2 gene amplification; ErbB2 is a key mediator of tumor cell growth and survival, and a predictor of poor outcome. Potential strategies to block peptide growth factor signaling pathways include (1) blocking or neutralizing the growth factor or its production, (2) blocking the receptor-binding site or prevention of receptor dimerization, (3) blocking the receptor tyrosine kinase domain, and (4) blocking downstream signaling pathways. The mechanism of action of trastuzumab is multifactorial and includes (1) antibody-dependent cellular cytotoxity, (2) modulation of ErbB2 signaling, (3) internalization of ErbB2 cell-surface protein, (4) reduction in proteolytic cleavage of the ErbB2 extracellular domain, and (5) interference with DNA damage repair. Small-molecule inhibitors such as lapatinib have an advantage over antibodies because they inhibit multiple signaling pathways at plasma concentrations achieved in the clinic. This is relevant because the survival of epithelial tumors is often dependent on the presence of redundant signaling pathways. Combining anti-ErbB2 antibodies with small-molecule tyrosine kinase inhibitors is an appealing strategy because both therapies exert their antitumor effects via distinct mechanisms of action and target ErbB2 at different sites. Combining distinct classes of inhibitors may not only potentiate cellular cytotoxicity, but may also assist in overcoming inherent or acquired resistance to one class of inhibitors. Mechanisms of resistance identified to date include activating mutations in the domain of the adenosine triphosphate–binding pocket for small-molecule tyrosine kinase inhibitors and the presence of truncated receptors for monoclonal antibodies. Dual-agent molecular targeting has been examined in preclinical models using the ErbB1 pathway as well as the ErbB2 pathway. With respect to the ErbB1 pathway, Huang et al and Matar et al studied the effect of cetuximab (an antibody that binds to the extracellular domain of ErbB1) in combination with either gefitinib or erlotinib (small-molecule inhibitors of the tyrosine kinase domain or ErbB1) against a panel of human cancer cell lines in culture and in tumor xenografts. The combination of cetuximab plus gefitinib or erlotinib enhances growth inhibition, in culture and in vivo, over that observed with either agent alone. Phosphorylation inhibition of downstream effector molecules (mitogen-activated protein kinase and AKT), and apoptosis was enhanced with the combination. In lung and head and neck tumor cells highly resistant to cetuximab, gefitinib, and erlotinib, but not cetuximab, retained the capacity to inhibit these tumor cells and were also able to further inhibit the activation of downstream effectors of ErbB1 signaling. When tested in vivo, single-agent therapy resulted in transient tumor regression only at the highest doses, whereas suboptimal doses of gefitinib and cetuximab in combination resulted in complete and permanent regression of large tumors. Contradictory results were obtained by Fishel et al who determined that gefitinib and cetuximab, when used in combination, were antagonistic when tested against two ErbB1expressing epidermoid cell lines. The combination caused less apoptosis than gefitinib alone, and this appeared to be the result of overexpression of functional ErbB1 associated with the combination. It is likely that these discordant results were a result of the particular cell lines studied. Most relevant to the study being reviewed are the preclinical models examining dual targeting of the ErbB2 pathway. Xia et al treated ErbB2-overexpressing breast cancer cell lines with a combination of lapatinib and either trastuzumab or a polyclonal anti-ErbB2 antibody. The combination treatments led to enhanced tumor cell apoptosis, whereas treatment with the same doses of lapatinib, trastuzumab, or polyclonal anti-ErbB2 antibody alone led to minimal or no increase in apoptosis. They demonstrated that the enhanced tumor cell apoptosis after combination ErbB2-targeted therapies was most closely associated with downregulation of survivin protein. Konecny et al studied the combination of lapatinib and trastuzumab in a panel of human breast cancer cell lines. Synergistic activity was seen when the combination was tested in ErbB2-overexpressing cell lines. Of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 20 JULY 1

EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: one-third of doublets occur at five pairs of amino acids

Doublet mutations in cancer are not well studied. We find that allelic somatic doublet mutations are present at high frequency in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain in lung cancers. When doublets from the literature are added, a total of 96 doublets are available for analysis. The frequency of doublets overall is 6%, which is sevenfold greater than that observed in normal tissue in mouse. All characterized doublets are allelic, and silent mutations occur rarely. About half of all doublets contain one or two of 12 distinct missense mutations at five amino acids: E709, G719, S768, T790 and L861. The mutations at these five amino acids are seldom reported as singlets. Moreover, when the common L858 target is included, more than one-third of EGFR doublets are one of five specific missense pairs: G719/E709, G719/S768, G719/L861, L858/E709 and L858/T790. Structure suggests function: The data imply that most EGFR doublets are NOT consistent with a 'driver and passenger' mutation mechanism. EGFR doublets are highly skewed relative to singlets, consistent with functional selection of two individually suboptimal mutations that, in combination, have enhanced oncogenic potential.

Spatial Structure of the Dimeric Transmembrane Domain of the Growth Factor Receptor ErbB2 Presumably Corresponding to the Receptor Active State

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

2007 Annual Meeting of the National Lung Cancer Partnership: A Summary of Meeting Highlights

This report presents highlights from The National Lung Cancer Partnership's Annual Meeting, held in June 2007 in Chicago. It discusses recent refinements in the histologic, genetic, and epigenetic subtyping of lung cancers and suggests reasons why certain therapies benefit only a subset of lung cancer patients. It also describes new molecular data about the subtype-specific differences in drug resistance among bronchioloalveolar-associated non-small cell lung cancers and discusses strategies to avoid or tackle specific drug-resistant tumors. Finally, it describes new findings about epigenetic differences-specifically in DNA hypermethylation-among lung tumors, including some male/female differences, which may prove useful as biomarkers for diagnosis, prognosis, and prediction of response to treatments.

Template‐directed Assembly of Signaling Proteins: A Novel Drug Screening and Research Tool

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer. However, a significant impediment to researchers remains: membrane-associated proteins are difficult to reconstitute and study. Template-directed assembly represents a powerful new technology that enables the assembly of membrane-associated proteins. We show that template-directed assembly restores tyrosine kinase activity and regulation, and provides a way for researchers to build multicomponent assemblies. As an example of better enzyme regulation, the Tie2 tyrosine kinase domain exhibits (biologically relevant) autoinhibitory behavior when template assembled. Also, template-assembled insulin receptor tyrosine kinase domains exhibit significant autophosphorylation (none detected without template-directed assembly) and an eightfold increase in substrate phosphorylation (compared to best solution conditions). Thus, template-directed assembly has a demonstrated ability to effectively produce more biologically relevant results using these commercial reagents. Template-directed assembly promises to be generally applicable to the signaling networks important for human health, because these pathways frequently contain membrane-associated proteins that require the organizing influence of a membrane surface.

Roles for deregulated receptor tyrosine kinases and their downstream signaling molecules in hematologic malignancies

Growth, survival and differentiation of hematopoietic cells are regulated by the interactions between hematopoietic growth factors and their receptors. The defect in these interactions results in a failure of hematopoiesis, while aberrantly elevated and/or sustained activation of these signals cause hematologic malignancies. Among them, constitutively activating mutations of the receptor tyrosine kinases (RTKs), such as c-Kit, platelet-derived growth factor receptor (PDGFR) and FLT3, are often involved in the pathogenesis of various types of hematologic malignancies. Constitutive activation of RTKs is provoked by several mechanisms including chromosomal translocations and various mutations involving their regulatory regions. Chromosomal translocations commonly generate chimeric proteins consisting of the cytoplasmic domain of RTKs and the dimerization or multimerization motif of the fusion partner, resulting in the constitutive dimerization of RTKs. On the other hand, missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop, and extracellular domain, also cause constitutive activation of RTKs mainly by preventing the auto-inhibitory regulation. Oncogenic RTKs activate downstream signaling molecules such as Ras/MAPK, PI3-K/Akt/mTOR, and STATs as well as ligand-activated wild type RTKs. However, their signals are quantitatively and qualitatively different from wild type RTKs. Based on these findings, several agents that target oncogenic RTKs or their downstream molecules have been developed: imatinib and FLT3 inhibitors for RTKs themselves, farnesyltransferase inhibitors, mTOR inhibitors and MEK inhibitors for the downstream signaling molecules. As promising results have been obtained in several clinical trials using these agents, the establishment of these molecular targeted agents is expected.

Establishment of a model to assess the clinical efficacy of Gefitinib in treatment of non-small cell lung cancer

To establish a model to predict the clinical response of Gefitinib in non-small cell lung cancer (NSCLC). The clinical outcomes of 262 consecutive advanced NSCLC patients to oral treatment of gefitinib 250 mg daily in the past 4 years were reviewed. DNA sequencing was used to detect the mutations in the exons 18, 19, 20, and 21 of the epidermal growth factor receptor (EGFR) tyrosine kinase domain in 55 patients who had enough tumor tissues. The response rate and disease control rate of gefitinib in the advanced NSCLC patients were 30.1% and 78.6% respectively. The median progression free survival and median overall survival were 6.0 and 16.0 months respectively, while the 1, 2 and 3-year survival rates of the NSCLC patients were 60.8%, 35.6%, and 18.3% respectively. The clinical response rate of the patients with EGFR mutation was 50.0%, significantly higher than that of those patients without mutation (16.1%; P = 0.009). In the majority of patients Gefitinib was well tolerated, and the common adverse effects were skin rash and diarrhea. Based on the results of our patients, we try to establish a model which may predict the response of patients by logistic multivariate regression analysis. Patients being female aged under 65, with adenocarcinoma, not smoking, taxone-unresistant and with EGFR mutation were more sensitive to gifitinib. However, COX multivariate regression analysis showed that only the age, histology, smoke status and EGFR mutation were valuable in selection of sensitive patients. So, we set up the cut-off value at 2 in the model based on these 4 factors. The response rate of the patients with 2 or more scores was 34.2%, significantly higher than that of the patients with the scores < 2 (9.3%, P = 0.001). The sensitivity would be doubled when the score increases by one point. A simple clinical model based on the patients' age, histology, smoking status and EGFR mutation has been established and is useful to determine the efficacy of gifitinib in NSCLC patients.

Oncogenic Fusion Tyrosine Kinases as Molecular Targets for Anti-Cancer Therapy

Deregulated activation of protein tyrosine kinases (PTKs) is a frequent event underlying malignant transformation in many types of cancer. The formation of oncogenic fusion tyrosine kinases (FTKs) resulting from genomic rearrangements, represents a common mechanism by which kinases escape the strict controls that normally regulate their expression and activation. FTKs are typically composed of an N-terminal dimerisation domain, provided by the fusion partner protein, fused to the kinase domain of receptor or non-receptor tyrosine kinases (non-RTKs). Since FTKs do not contain extracellular domains, they share many characteristics with non-RTKs in terms of their properties and approaches for therapeutic targeting. FTKs are cytoplasmic or sometimes nuclear proteins, depending on the normal distribution of their fusion partner. FTKs no longer respond to ligand and are instead constitutively activated by dimerisation induced by the fusion partner. Unlike RTKs, FTKs cannot be targeted by therapeutic antibodies, instead they require agents that can cross the cell membrane as with non-RTKs. Here we review the PTKs known to be expressed as FTKs in cancer and the strategies for molecularly targeting these FTKs in anti-cancer therapy.

A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

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.

7502 ORAL Assessment of plasma levels of soluble KIT (sKIT) as a potential surrogate marker for TTP in patients (pts) with advanced gastrointestinal stromal tumor (GIST) treated with sunitinib

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.

Epidermal Growth Factor Receptors with Tyrosine Kinase Domain Mutations Exhibit Reduced Cbl Association, Poor Ubiquitylation, and Down-regulation but Are Efficiently Internalized

Some non-small cell lung cancers (NSCLC) with epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations require altered signaling through the EGFR for cell survival and are exquisitely sensitive to tyrosine kinase inhibitors. EGFR down-regulation was impaired in two NSCLCs with EGFR tyrosine kinase domain mutations. The mutant receptors were poorly ubiquitylated and exhibited decreased association with the ubiquitin ligase Cbl. Overexpression of Cbl increased the degradation of EGFR. Treatment with geldanamycin, an inhibitor of the chaperone heat shock protein 90, also increased both wild-type and mutant EGFR degradation without affecting internalization. The down-regulation of the mutant EGFRs was still impaired when they were stably expressed in normal human bronchial epithelial cells. Thus, the mutations that altered signaling also decreased the interaction of EGFRs with the mechanisms responsible for endosomal sorting.

P2-044: Lapatinib increases cytotoxicity against gefitinib-resistant T790M lung cancer cells by inhibiting active heterodimerization of EGFR and HER-2

Somatic mutations in epidermal growth factor receptor (EGFR) tyrosine kinase domain predicts the response to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). The emergence of acquired resistance to EGFR TKI has been recently described in patients whose tumor initially responded to gefitinib. Lapatinib is a dual inhibitor of ErbB1 (EGFR) and ErbB2 (HER-2) tyrosine kinases and has shown promising efficacy in HER-2 overexpressing breast cancer. However, its therapeutic role is not fully studied in NSCLC. Here we report that lapatinib enhances cytotoxicity against gefitinib-resistant NSCLC by blocking active heterodimerization of EGFR and HER-2. We identified a secondary T790M mutation in a NSCLC tumor with a gefitinib-sensitive L858R mutation that eventually progressed after initial response. Acquired T790M mutation conferred resistance to L858R mutant cells sensitive to gefitinib. Of the various molecules downstream of EGFR, signal transduction and activator of transcription 3 (Stat3) signaling was continuously activated in L858R/T790M cells, and the inhibition of Stat3 suppressed gefitinib-resistant cell growth. As for Stat3 inhibition, we found that lapatinib inactivated Stat3 and inhibited cell growth in gefitinib-resistant NSCLC. Finally, lapatinib was also found to block the heterodimerization of EGFR and HER-2, and this led to the inactivation of Stat3 in gefitinib-resistant T790M cells, whereas the active heterodimerization of EGFR and HER-2 was maintained when these cells were treated with gefitinib. Taken together, our data suggest that lapatinib may overcome gefitinib resistance in NSCLC. A clinical investigation of the use of lapatinib in gefitinib-resistant NSCLC is strongly warranted.

Novel and existing mutations in the EGF receptor tyrosine kinase domain are predictors of optimal resectability (OR) in malignant peritoneal mesothelioma (MPM)

10503 Background: MPMs represent 20% of all malignant mesothelioma (MM) cases. The median survival for these tumors is less than a year, and like other peritoneal tumors this is due to intra-abdominal recurrence/progression. Currently there is a paucity of information about the biology of these tumors and molecular perturbations that are involved in tumor formation. Elucidation of mutations and biological pathways active in these tumors may identify valuable prognostic markers, as well as facilitate the development of novel therapies. Methods: Twenty-nine MPM patients were evaluated at a single tertiary center and their tumors were probed for mutations in the catalytic TK domain of EGF receptor (mut+). All specimens were examined for somatic mutations by PCR amplification. Twenty-five patients were treated with cytoreductive surgery (CRS) ± IPHC and complete clinical data including age, sex, cytoreductive score, histology, mutation, and survival was available for comparison of the mut+ and mut- groups. Results: The median age was 56, and 71% of the patients were male with a median follow-up time (MFT) of 14.5 months. Mutations were found in 31% (9/29) of the tumors, 7 of these mutations were novel, and the 8th one was the L858R mutation described in NSCLC. Of the 25 patients managed surgically there were 7 mut+ and 18 mut- ( Table ). OR (=R2a) was achieved in 7/7 (100%) of mut+ group and 9/18 (50%) mut- (p=.03). All mut+ are alive with a MFT of 24 months, while 5/18 (28%) of the mut- group are DOD with a MFT of 7 months (p=.27). In an ANCOVA model OR (p=0.04) was found to be predictive of survival. Conclusions: The EGFR-TK appears to be a common site for mutation in MPM, with the identification of novel and known activation mutation in 31% of tumors. Mut+ was predictive of OR. OR was predictive of survival in short term follow-up, and with longer follow-up, mut+ may be predictive of survival as well as represent a subset of patients who may be responsive to TK-inhibitors therapy. [Table: see text] No significant financial relationships to disclose.

Generation of intracellular domain of insulin receptor tyrosine kinase by γ-secretase

The proteolytic cleavage of a precursor protein into α- and β-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/γ-secretase-dependent processing. In cells treated with γ-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/γ-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/γ-secretase-dependent processing. We also show that the autophosphorylation levels of the IR β-subunit upon insulin stimulation were decreased by the inactivation of PS/γ-secretase, raising the possibility that the PS/γ-secretase proteolysis of IR may play a modulatory role in insulin signaling.

α-Lipoic Acid as a Directly Binding Activator of the Insulin Receptor: Protection from Hepatocyte Apoptosis

Alpha-lipoic acid has cytoprotective potential which has previously been explained by its antioxidant properties. The aim of this study was to assess LA-induced-specific cytoprotective signalling pathways in hepatocytes. Apoptosis of rat hepatocytes was induced by actinomycinD/TNF-alpha. Caspase-3-like activity was determined by a fluorometric; LDH by an enzymatic assay; and phosphorylation of the insulin receptor, Akt, and Bad by Western blot (after immunoprecipitation). Protein kinase and insulin receptor activities were measured by in vitro phosphorylation. Computer modeling studies were performed by using the program GRID. Alpha-lipoic acid decreased actinomycinD/TNF-alpha-induced apoptosis, as did the antioxidants Trolox and N-acetylcysteine. The activation of PI3-kinase/Akt involving phosphorlyation of Bad markedly contributed to the cytoprotective action of alpha-lipoic acid. Alpha-lipoic acid but not other antioxidants protected against actinomycinD/TNF-alpha-induced apoptosis via phosphorylation of the insulin receptor. Computer modeling studies revealed a direct binding site for alpha-lipoic acid at the tyrosine kinase domain of the insulin receptor, suggesting a stabilizing function in loop A that is involved in ATP binding. Treatment of immunoprecipitated insulin receptor with LA induced substrate phosphorylation. Alpha-lipoic acid mediates its antiapoptotic action via activation of the insulin receptor/PI3-kinase/Akt pathway. We show for the first time a direct binding site for alpha-lipoic acid at the insulin receptor tyrosine kinase domain, which might make alpha-lipoic acid a model substance for the development of insulin mimetics.

Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.