Receptor Tyrosine Kinase Domain Research Articles

P3.03-006 Optical Control of Growth Factor Receptors to Advance Signal Transduction Research and Drug Screening: Topic: Mesothelioma Transitional

Growth factor receptors are central elements of signal transduction pathways and increasingly important targets for anticancer drugs. In recent years naturally occurring light sensitive protein domains (LSPDs) from different kingdoms of life have been used to generate genetically encoded chimeric signaling molecules that can be activated reversibly and with spatiotemporal precision by light. The development of such optogenetic tools has led to a plethora of new discoveries in the neurosciences but has received comparably little attention in cancer research — partly due to a lack of appropriate tools. Our aim was therefore to generate synthetic growth factor receptors that can be activated with light and allow fine-tuned control of growth factor-associated signal transduction pathways. To generate receptor tyrosine kinases (RTKs) that can be optically activated (Opto-RTKs), intracellular domains of RTKs were fused to LSPDs of the light-oxygen voltage (LOV) family from various species. The resulting chimeric receptors were tested for light-dependent activation of signal transduction by reporter gene assays, immunoblotting and various cell biological tests assessing DNA synthesis, epithelial mesenchymal transition (EMT) and angiogenesis. Three of the tested LOV domains enabled light-dependent receptor dimerization and activation of the corresponding signal transduction pathways when fused to the intracellular domains of FGFR1, EGFR, RET, c-Met or ROS1. Opto-RTKs enabled stringent control of the MAPK, PI3K and PLCγ pathways. Signal activation could be spatially confined to illuminated regions of culture plates and signals rapidly subsided after cessation of illumination. Light was able to replace FGF2 for the induction of cell proliferation and EMT in mesothelioma cells and VEGF for the stimulation of angiogenic sprouting in endothelial cells. Moreover, Opto-RTKs enabled light-assisted screening for small molecule inhibitors of EGFR, FGFR1 and the orphan RTK ROS1. Our optogenetic approach allows light-mediated control of growth factor receptors representing clinically relevant drug targets. Opto-RTKs enable dissection of dynamic signals with increased spatiotemporal resolution and open new possibilities for drug screening. Transfer of the design principle to additional membrane receptors is ongoing.

Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.

Abstract 3257: The impact of dual targeting VEGFR2 and Tie-2 on angiogenesis using in vitro model

Abstract Angiogenesis is an essential key process in tumor growth, invasion, and metastasis. Several angiogenesis inhibitors have been clinically proven to treat a wide variety of cancer types. These agents target either the vascular endothelial growth factor (VEGF) or the intracellular tyrosine kinase domain of VEGF receptors. Despite the encouraging clinical benefits, there are multiple challenges such as significant toxic effects and rapid development of drug resistance which limit the long-term benefits of VEGF-targeted therapy. Emerging evidence suggests that tumors can bypass VEGF/VEGFR inhibition by up-regulating other pro-angiogenic pathways to compensate for VEGF inhibition. For these reasons, additional strategies to inhibit tumor vasculature are being sought. In this regard, F16, a new compound developed by Rumbaugh-Goodwin Institute for Cancer Research, harbors the potential for suppressing VEGF-driven angiogenesis by selectively blocking the extracellular domain of VEGFR2, which plays a crucial role in endothelial cell proliferation, survival, and migration. F16 has shown a remarkable efficacy in inhibiting VEGFR-associated tyrosine kinase activities. On the other hand, Tie-2 receptor is predominantly expressed in the endothelium and has been shown to facilitate VEGF-mediated angiogenesis. Therefore, we hypothesize that dual targeting of VEGFR2 and Tie-2 using F16 and T2i respectively will result in augmented angiogenesis inhibition. To test our hypothesis, human umbilical vein endothelial cells (HUVECs) were used as a model for our initial validation studies. Our preliminary data indicated that combining F16 and T2i significantly inhibited HUVECs proliferation compared to monotherapies (P<0.001) and 90% inhibition of HUVECs proliferation was observed at 1μM of the drug combination. Moreover, the ability of the indicated drugs to inhibit angiogenesis was further evaluated using a Matrigel-based tube formation assay. Using the scoring system developed by our laboratory, combining F16 and T2i at 1μM scored five pluses, indicating that individual cells were well-separated as compared to monotherapies (three pluses and two pluses respectively). These findings clearly indicated that concurrent blockade of VEGFR2 and Tie-2 represented a new strategy to potentiate angiogenesis inhibition. However, further studies are required to test whether there would be imposing changes in the growth rates of human cancers implanted in a mouse model, and whether these changes are attributed to changes in tumor vasculatures. These studies will ultimately lead us to understand the pharmacodynamics of F16 and Tie-2 inhibitor for better clinical applications in the future. (This research was supported by Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida.) Citation Format: Khalid Alhazzani, Thanigaivelan Kanagasabai, Saad Alobid, Appu Rathinavelu. The impact of dual targeting VEGFR2 and Tie-2 on angiogenesis using in vitro model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3257.

Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer – Proof of concept towards a systems pharmacology approach

Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006rERLO0.5 adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006rERLO0.5 cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006rERLO0.5 cells. Taken together, differential proteins in the EGFR interactome of HCC4006rERLO0.5 cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance.

NMR Experiments on Wild-Type and Mutant Fibroblast Growth Factor Receptor Kinases Reveal Conformational Dynamics Associated with Enzyme Activation

The kinase domain of receptor tyrosine kinases (RTKs) is responsible for phosphorylating intracellular substrates upon hormone binding to the extracellular ectodomain. The resulting signal cascade is essential for several biological processes in human biology, including development and metabolism. Mutations in the kinase domain of RTKs can enhance the intrinsic activity of the enzyme and over-stimulate the resulting signal cascade leading to a wide variety of cancers and growth defects. Although a plethora of kinase crystal structures have been resolved, the dynamics associated with pathogenic mutations remain poorly understood. Here we analyzed the dynamics of pathogenic mutations within the fibroblast growth factor receptor kinase domain (FGFRK) as probed using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. Previous work showed that mutations at a conserved lysine residue in the activation loop bypassed the need for activation loop phosphorylation by shifting the population of the kinase from an autoinhibited state toward an active conformation. Interestingly, the degree of active state directly correlated with the kinase activity both in vitro and in human disease. The most prominent gain-of-function mutant was a lysine to glutamic acid substitution, which was found to stabilize the active conformation of the activation loop without the need for tyrosine phosphorylation (i.e., normal activation). The observed dispersions acquired on these pathogenic kinases using the CPMG experiments reveal conformational dynamics of the kinase in switching between active and inactive conformations and thus enabled us along with kinase crystal structures to identify an allosteric pathway spanning from the activation loop to the kinase hinge. These data agree well with the structural differences observed between kinases harboring different activating mutations, thus explaining how intrinsically activating mutations propagate their effects through the kinase.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mutational analysis of the insulin-like growth factor 1 receptor tyrosine kinase domain in non-small cell lung cancer patients.

The insulin-like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well-documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single-nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16-21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease-free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.

An In silico Chimeric Vaccine Targeting Breast Cancer Containing Inherent Adjuvant.

Background:Today, Lack of efficient therapeutic strategy for breast cancer (the most common cause of death in women) is one of the momentous problematic topics for all health care committees. Designing new specific vaccine, based on antigens located on the surface of cancer cells can be useful. Over expression of ROR1, lacked of HER2/neu, and hormone receptors on cell surface in the breast cancer, introduce this protein as an appropriate candidate for designing cancer vaccine. Objectives:We hypothesized the extracellular domain of receptor tyrosine kinase like orphan receptor 1 (ROR-1) along with a super antigen such as staphylococcal enterotoxin B could be a potent vaccine for drug resistant breast cancer.Materials and Methods:Here, we assessed the findings of bioinformatics analysis to identify the antitumor immune properties of this chimeric construct. In addition, the stability, physic-chemical properties and allergic potency of designed fusion protein were investigated by valid bioinformatics software.Results:Our result suggested that chimeric model is capable to be a stimulant of both T-cell and B- cell mediated immune responses with an acceptable accessibility and solubility but without any allergenicity. Conclusions:The ROR-1 with an enterotoxin B could be a potent vaccine for breast cancer.

Activation and Decay Kinetics of the RET Receptor Tyrosine Kinase

We use ZD6474, an inhibitor of the kinase domain of the RET receptor tyrosine kinase, as a chemical probe to obtain quantitative information on the molecular kinetics of RET activation, autophosphorylation, dephosphorylation and trafficking in mouse neuroblastoma cells. Stimulation with RET's cognate ligand artemin (ART) resulted in up to 65% of cellular RET becoming phosphorylated over several minutes, followed by a slow decay. Addition of ZD6474 6 min after stimulation resulted in loss of phosphoRET (pRET) with t1/2 蠄 1 min, indicating that overall pRET dynamics reflects an evolving steady state between rapid RET phosphorylation and its comparably rapid dephosphorylation. Reconciling the observed rate of pRET formation with the rapid rate of pRET dephosphorylation and the high level of pRET at the steady state required invoking a rate-limiting activation of RET by ART, prior to receptor phosphorylation which occurs rapidly (rate constant 1.2 min-1). Dephosphorylation of pRET regenerates activated but unphosphorylated RET, which autophosphorylates again rapidly rather than reverting to the resting state. We additionally show that this rapidly cycling pRET is rapidly internalized through clathrin-coated pits, where it continues to activate ERK and Akt signaling. A fraction of the internalized pRET slowly converts to a longer-lived form (t1/2 ~ 10 min) that resides in late endosomes where it is protected from phosphatases and loses its signaling ability. Our results kinetically distinguish ART-dependent assembly of the activated RET receptor complex from its subsequent autophosphorylation, and provide a quantitative picture of RET receptor activation and trafficking.

Class III Receptor Tyrosine Kinases in Acute Leukemia - Biological Functions and Modern Laboratory Analysis.

Acute myeloid leukemia (AML) is a complex disease caused by deregulation of multiple signaling pathways. Mutations in class III receptor tyrosine kinases (RTKs) have been implicated in alteration of cell signals concerning the growth and differentiation of leukemic cells. Point mutations, insertions, or deletions of RTKs as well as chromosomal translocations induce constitutive activation of the receptor, leading to uncontrolled proliferation of undifferentiated myeloid blasts. Aberrations can occur in all domains of RTKs causing either the ligand-independent activation or mimicking the activated conformation. The World Health Organization recommended including RTK mutations in the AML classification since their detection in routine laboratory diagnostics is a major factor for prognostic stratification of patients. Polymerase chain reaction (PCR)–based methods are well-validated for the detection of fms-related tyrosine kinase 3 (FLT3) mutations and can easily be applied for other RTKs. However, when methodological limitations are reached, accessory techniques can be applied. For a higher resolution and more quantitative approach compared to agarose gel electrophoresis, PCR fragments can be separated by capillary electrophoresis. Furthermore, high-resolution melting and denaturing high-pressure liquid chromatography are reliable presequencing screening methods that reduce the sample amount for Sanger sequencing. Because traditional DNA sequencing is time-consuming, next-generation sequencing (NGS) is an innovative modern possibility to analyze a high amount of samples simultaneously in a short period of time. At present, standardized procedures for NGS are not established, but when this barrier is resolved, it will provide a new platform for rapid and reliable laboratory diagnostic of RTK mutations in patients with AML. In this article, the biological and physiological role of RTK mutations in AML as well as possible laboratory methods for their detection will be reviewed.

Label-Free Characterization of a Novel Early-Stage Drug Discovery Platform

Template-Directed Assembly (TDA) of membrane-associated proteins is a discovery tool that has been in use for some time. There is an abundance of data that suggests that recombinant proteins cloned without their transmembrane or membrane anchoring domains do not behave as they do in the cell. TDA technology utilizes the common affinity tag (multi-histidine) to assemble recombinant fragments of membrane targets (single pass cytoplasmic or extracellular) onto liposomes incorporating Ni-chelating headgroups. In many cases, these assembled recombinant proteins demonstrate cell-like activity and selectivity. Despite the abundance of activity data that exists, there is a relative paucity of biophysical characterization of these systems. Importantly, it has been proposed that TDA-assembled molecules are conformationally distinct from the unassembled cloned fragments and more cell-like in character. Using differential scanning calorimetry (DSC), we present data comparing the thermal stability of TDA-assembled cytoplasmic domains of transmembrane receptor tyrosine kinases to unassembled RTK domains. We also compare thermal stability of the RTK domains in conditions previously described to promote maximum activity (with Mn as counterion instead of Mg). Using Dynamic Light Scattering (DLS), we will characterize the size of assembled complexes and probe the aggregation state of RTK fragments under the conditions described above. Data will be assessed and correlated with activity data.

Biomarkers for predicting response to tyrosine kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells.

The epidermal growth factor receptor (EGFR) family is reportedly overexpressed in bladder cancer, and tyrosine kinase inhibitors (TKIs) have been suggested as treatment. Gefitinib (Iressa®) is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor). Both compounds compete with the binding of ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical issue. The purpose of the present study was to use protein array analysis to compare the signaling pathway(s) induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive) and UM-UC-14 (drug-resistant) bladder cancer cells and to identify molecular markers that may be useful predictors of their efficacy. The results revealed that phosphorylation of EGFR, HER3, Met and ERK1/2 was markedly overexpressed in the sensitive cell line (UM-UC-5) and was strongly inhibited by the TKIs. Other notable differences included decreased phosphorylation of RSK, GSK3, AMPK, Akt and c-Jun by TKIs in the sensitive cells. In contrast, phosphorylated p53 was highly expressed in the resistant cell line (UM-UC-14) and TKIs had no effect in the resistant cells. Overall results suggest that phosphorylated HER3, ERK1/2 and p53 may be used as biomarkers to determine the sensitivity of bladder cancers to TKIs. In particular, a combination of these markers may be more likely to predict the sensitivity to TKIs.

The role of AXL and the in vitro activity of the receptor tyrosine kinase inhibitor BGB324 in Ewing sarcoma.

New targets for Ewing sarcoma (ES) patients are urgently needed. Therefore, we investigated the expression and genetic aberrations of the oncogenic receptor tyrosine kinase (RTK) AXL in ES and determined the efficacy of AXL targeting on cell viability and migration. First, AXL and Gas6 (ligand) mRNA expression was determined by RT-PCR on 29 ES samples. Low, medium and high AXL mRNA expression was observed in 31% (n = 9), 48% (n = 14) and 21% (n = 6) of samples. Gas6 was abundantly present in all specimens. We next tested AXL protein expression immunohistochemically in 36 tumors (primary, post-chemotherapy, metastasized and relapsed samples) from 25 ES patients. Low, medium and high AXL protein expression was observed in 17% (n = 6), 19% (n = 7) and 36% (n = 13) of samples. In primary tumors (n = 15), high AXL expression correlated significantly with a worse overall survival compared to patients with lower expression (61 vs. 194 months, p = 0.026). No genetic aberrations were detected in the AXL RTK domain (n = 29). The AXL-inhibitor BGB324 affected viability (IC50 0.79-2.13 μmol/L) and migratory potential of all tested ES cell lines in vitro (n = 5-6). BGB324 chemosensitized chemotherapy-resistant ES-4 cells to vincristine and doxorubicin. These data suggest that AXL is a potential novel, druggable therapeutic target in ES.

Insulin resistance induced by hyperinsulinemia coincides with a persistent alteration at the insulin receptor tyrosine kinase domain.

Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response. If so, insulin resistance would worsen as the level of circulating insulin increases during compensation, which could contribute to the transition of insulin resistance to more severe disease. Here, we investigated the consequences of excess insulin exposure to insulin receptor (IR) activity. Cells chronically exposed to insulin show a diminished the level of IR tyrosine and serine autophosphorylation below that observed after short-term insulin exposure. The diminished IR response did not originate with IR internalization since IR amounts at the cell membrane were similar after short- and long-term insulin incubation. Förster resonance energy transfer between fluorophores attached to the IR tyrosine kinase (TK) domain showed that a change in the TK domain occurred upon prolonged, but not short-term, insulin exposure. Even though the altered ‘insulin refractory’ IR TK FRET and IR autophosphorylation levels returned to baseline (non-stimulated) levels after wash-out of the original insulin stimulus, subsequent short-term exposure to insulin caused immediate re-establishment of the insulin-refractory levels. This suggests that some cell-based ‘memory’ of chronic hyperinsulinemic exposure acts directly at the IR. An improved understanding of that memory may help define interventions to reset the IR to full insulin responsiveness and impede the progression of insulin resistance to more severe disease states.

The Role of Axl in Ewing Sarcoma

ABSTRACT Aim: Ewing sarcoma (ES) is an aggressive form of primary bone cancer affecting mainly children and young adults. Despite intensive multimodal treatment, survival has not improved much during the last decade and therapeutic options in second line are scarce. Therefore, the search for novel, targeted therapeutic strategies has a high priority. Here, we investigated for the first time the expression and genetic aberrations of AXL, an oncogenic receptor tyrosine kinase (RTK), in ES and determined the efficacy of AXL-targeting on cell viability and migration in vitro. Methods: AXL and Gas6 (ligand) mRNA expression were determined by RT-PCR on 29 fresh-frozen ES patient samples. AXL protein expression was determined immunohistochemically in 36 tumors (primary tumors, post-chemotherapy resections, metastases and relapses) from 25 ES patients. Kaplan-Meier analysis (log-rank test) was performed to draw and evaluate the significance of survival curves. The AXL RTK domain and Gas6 were sequenced in 29 ES tumors. Six ES cell lines (ES1, ES2, ES4, ES7, ES8 and EW8) were treated in vitro with the specific AXL-inhibitor BGB324 (R428) and viability was analyzed with MTT-assays. In vitro migration assays were performed to investigate effects of BGB324 on ES cell migration. Results: Low, medium and high AXL mRNA expression was observed in 31% (n=9), 48% (n=14) and 21% (n=6) of ES samples, respectively. Gas6 was abundantly present in all specimens. Low, medium and high AXL protein expression was observed in 16% (n=6), 18% (n=7) and 34% (n=13) of ES samples. In primary tumors (n=15), high AXL expression correlated significantly with a worse overall survival (OS) compared to patients with lower expression (61 vs. 194 months, p=0.026). No genetic aberrations were observed in the AXL RTK domain or Gas6. BGB324 dose-dependently affected ES cell viability (IC50 0.79 - 2.13 mmol/L) and migratory potential (∼2 times delayed wound closure) in vitro. Conclusions: Our data suggest that AXL is a potential novel therapeutic target in ES. We are currently examining the effects of BGB324 on human derived ES xenografts in vivo. Because AXL-targeting drugs, such as BGB324, entered the phase of clinical trials, AXL targeting may potentially be of interest for further exploration in the clinical ES setting. Disclosure: All authors have declared no conflicts of interest.

Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr(921), Tyr(930), and Tyr(960), has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr(921) and Tyr(930) enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.

Luteolin is effective in the non‐small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed malignancies in the world. Small-molecule inhibitors of the EGF receptor's tyrosine kinase domain (TKIs), including gefitinib and erlotinib, have been widely used for treating NSCLC. Unfortunately, nearly all patients after initially experiencing a marked improvement while on these drugs, eventually progress to acquire resistance to TKIs. Because there is no effective therapeutic strategy to treat TKI-resistant NSCLC, we evaluated the effects of luteolin, a naturally occurring flavanoid, on T790M mutant NSCLC cells. The effect of luteolin on the viability of NSCLC and normal cell lines was investigated using the Cell Counting Kit-8 (CCK-8) assay. Luteolin-induced apoptosis was assessed by bivariate FITC-annexin V/PI assay, and Western blots were used to measured apoptotic proteins. Co-immunoprecipitation was used to determine the effect of luteolin on the interaction between Hsp90 and mutant EGF receptors. The effect of luteolin on the Akt/mTOR pathway was studied using Western blotting analysis. Its anti-tumour efficacy in vivo was examined in a mouse xenograft model. Luteolin exerted significant anti-tumourigenic effects on the EGF receptor L858R/T790M mutation and erlotinib-resistant NSCLC both at the cellular and animal levels. Mechanistically, luteolin induced degradation of the EGF receptor by inhibiting the association of Hsp90 with the mutant EGF receptor, and, therefore, prevented PI3K/Akt/mTOR signalling, which resulted in NSCLC cell apoptosis. Luteolin may be a potential candidate for NSCLC therapy, especially for treatment of patients with acquired erlotinib-resistant NSCLC.

Identification of a Src kinase SH3 binding site in the C‐terminal domain of the human ErbB2 receptor tyrosine kinase

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.

Computational delineation of tyrosyl-substrate recognition and catalytic landscapes by the epidermal growth factor receptor tyrosine kinase domain.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK), which catalyzes protein phosphorylation reactions by transferring the γ-phosphoryl group from an ATP molecule to the hydroxyl group of tyrosine residues in protein substrates. EGFR is an important drug target in the treatment of cancers and a better understanding of the receptor function is critical to discern cancer mechanisms. We employ a suite of molecular simulation methods to explore the mechanism of substrate recognition and to delineate the catalytic landscape of the phosphoryl transfer reaction. Based on our results, we propose that a highly conserved region corresponding to Val852-Pro853-Ile854-Lys855-Trp856 in the EGFR tyrosine kinase domain (TKD) is essential for substrate binding. We also provide a possible explanation for the established experimental observation that protein tyrosine kinases (including EGFR) select substrates with a glutamic acid at the P - 1 position and a large hydrophobic amino acid at the P + 1 position. Furthermore, our mixed quantum mechanics/molecular mechanics (QM/MM) simulations show that the EGFR protein kinase favors the dissociative mechanism, although an alternative channel through the formation of an associative transition state is also possible. Our simulations establish some key molecular rules in the operation for substrate-recognition and for phosphoryl transfer in the EGFR TKD.

Molecular Origin of the Binding of WWOX Tumor Suppressor to ErbB4 Receptor Tyrosine Kinase

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in downregulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, nonconsensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of the WW1 domain of WWOX. Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX in agreement with our findings reported previously. Altogether, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes.