Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor

The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-A resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva(TM)). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.     

The insulin and EGF receptor structures: new insights into ligand-induced receptor activation

The insulin receptor (IR) and epidermal growth factor receptor (EGFR; also known as ErbB) families exhibit similarities in the composition of their ectodomains. The past five years have seen structures determined for all members of the EGFR family including some complexes with ligand or monoclonal antibody fragments. These structures have led to a clearer understanding of their mechanism of activation and inhibition. By contrast, obtaining equivalent understanding of the IR family has lagged behind. However, within the past year, structures of partial and complete ectodomains of the IR have been published that show that the extracellular region of the receptor adopts an unexpected 'inverted V' conformation relative to the cell membrane. This is very different from the folded-over (tethered) conformation of the unactivated EGFR and provides insight into the potential mechanism of activation of the IR.     

NSP1 defines a novel family of adaptor proteins linking integrin and tyrosine kinase receptors to the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway

As part of a program to further understand the mechanism by which extracellular signals are coordinated and cell-specific outcomes are generated, we have cloned a novel class of related adaptor molecules (NSP1, NSP2, and NSP3) and have characterized in more detail one of the members, NSP1. NSP1 has an Shc-related SH2 domain and a putative proline/serine-rich SH3 interaction domain. Treatment of cells with epidermal growth factor or insulin leads to NSP1 phosphorylation and increased association with a hypophosphorylated adaptor protein, p130(Cas). In contrast, cell contact with fibronectin results in Cas phosphorylation and a transient dissociation of NSP1 from p130(Cas). Increased expression of NSP1 in 293 cells induces activation of JNK1, but not of ERK2. Consistent with this observation, NSP1 increases the activity of an AP-1-containing promoter. Thus, we have described a novel family of adaptor proteins, one of which may be involved in the process by which receptor tyrosine kinase and integrin receptors control the c-Jun N-terminal kinase/stress-activated protein kinase pathway.     

The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules

Background  

Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11–12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins.     

Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.     

The cellular response to neuregulins is governed by complex interactions of the erbB receptor family.

Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.     

Structure and function of the receptor-like protein kinases of higher plants

Cell surface receptors located in the plasma membrane have a prominent role in the initiation of cellular signalling. Recent evidence strongly suggests that plant cells carry cell surface receptors with intrinsic protein kinase activity. The plant receptor-like protein kinases (RLKs) are structurally related to the polypeptide growth factor receptors of animals which consist of a large extracytoplasmic domain, a single membrane spanning segment and a cytoplasmic domain of the protein kinase gene family. Most of the animal growth factor receptor protein kinases are tyrosine kinases; however, the plant RLKs all appear to be serine/threonine protein kinases. Based on structural similarities in their extracellular domains the RLKs fall into three categories: the S-domain class, related to the self-incompatibility locus glycoproteins of Brassica; the leucine-rich repeat class, containing a tandemly repeated motif that has been found in numerous proteins from a variety of eukaryotes; and a third class that has epidermal growth factor-like repeats. Distinct members of these putative receptors have been found in both monocytyledonous plants such as maize and in members of the dicotyledonous Brassicaceae. The diversity among plant RLKs, reflected in their structural and functional properties, has opened up a broad new area of investigation into cellular signalling in plants with far-reaching implications for the mechanisms by which plant cells perceive and respond to extracellular signals.     

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.     

Differential association of phosphatidylinositol-5-phosphate 4-kinase with the EGF/ErbB family of receptors.

Phosphatidylinositol-5-phosphate 4-kinase (PIP4K) is required for the production of phosphoinositol-4,5-hisphosphate (PIP2), which has been closely associated with growth factor signalling. Here we have tested the possibility that phosphoinositide kinases may be take part in signal transduction through interactions with the epidermal growth factor (EGF) receptor and the ErbB family of tyrosine kinase receptors. Interactions of the Type IIbeta isoform of PIP4K were observed with the EGF receptor family members in a number of diverse cell lines, including A431, PC12 and MCF7 cells but not with the N6F TrkA receptor. Co-immunoprecipitation experiments indicate that PIP4K interacts with not only the EGF receptor, but also selectively with members of the ErbB tyrosine kinase family. These results demonstrate another enzyme substrate for EGF receptors that facilitates the production of phosphoinositides at the cell membrane.     

Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.     

Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters beta-tubulin isotype expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Rat liver membranes contain a 120 kDa glycoprotein which serves as a substrate for the tyrosine kinases of the receptors for insulin and epidermal growth factor

The receptors for insulin and epidermal growth factor possess tyrosine-specific protein kinase activity which may play a role in mediating the biological actions of these two peptides. We have identified a 120 kDa glycoprotein (pp120) in rat liver plasma membranes which can be phosphorylated by the insulin receptor in a cell-free system and in intact cultured hepatoma cells. In the present report, we have demonstrated in a cell-free system that solubilized epidermal growth factor receptors can phosphorylate tyrosine residues in pp120.     

Adult rat otic placode-derived neurons and sensory epithelium express all four erbB receptors: a role in regulating vestibular ganglion neuron viability.

The erbB receptor family consists of erbB1/epidermal growth factor receptor, erbB2/neu, erbB3, and erbB4, all of which have been implicated in cell proliferation, differentiation, and survival in several tissues. In the nervous system, these family members can function in a trophic capacity for certain subpopulations of neurons and some types of non-neuronal cells. Vestibular sensory epithelial cells and vestibular ganglion neurons are derived from ectodermal otic placode and are essential components of the peripheral vestibular system, the sensory system for balance. Recent studies in mammals suggest that certain ligands of the epidermal growth factor receptor can induce proliferation of vestibular sensory epithelial cells. We now show that vestibular ganglion neurons and vestibular sensory epithelial cells express all four erbB receptors in adult rats. Cultured vestibular ganglion neurons also expressed all four erbB family members and were therefore used to analyze the effects of modulating erbB signaling on differentiated vestibular ganglion neurons. Transforming growth factor-alpha (a ligand for epidermal growth factor receptor) and sensory and motor neuron-derived factor (a ligand for erbB3 and erbB4) promoted vestibular ganglion neuron viability, whereas epidermal growth factor (another ligand for epidermal growth factor receptor) did not. Glial growth factor 2 (another ligand for erbB3 and erbB4) and an antibody that blocks erbB2/neu-mediated signaling inhibited vestibular ganglion neuron viability. Collectively, these observations indicate that erbB signaling regulates the viability of differentiated otic placode-derived cells in mammals and suggest that exogenous modulation of erbB signaling in peripheral vestibular tissues may prove therapeutically useful in peripheral vestibular disorders.     

A combination of two antibodies recognizing non‐overlapping epitopes of HER2 induces kinase activity‐dependent internalization of HER2

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down‐regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody‐induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non‐overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody‐induced HER2 down‐regulation.     

Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v-erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine-kinase receptor family.     

A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase.