An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.     

Epidermal growth factor receptors: function modulation by phosphorylation and glycosylation interplay

Post-translational modifications (PTMs) of proteins induce structural and functional changes that are most often transitory and difficult to follow and investigate in vivo. In silico prediction procedures for PTMs are very valuable to foresee and define such transitory changes responsible for the multifunctionality of proteins. Epidermal growth factor receptor (EGFR) is such a multifunctional transmembrane protein with intrinsic tyrosine kinase activity that is regulated primarily by ligand-stimulated transphosphorylation of dimerized receptors. In human EGFR, potential phosphorylation sites on Ser, Thr and Tyr residues including five autophosphorylation sites on Tyr were investigated using in silico procedures. In addition to phosphorylation, O-GlcNAc modifications and interplay between these two modifications was also predicted. The interplay of phosphorylation and O-GlcNAc modification on same or neighboring Ser/Thr residues is termed as Yin Yang hypothesis and the interplay sites are named as Yin Yang sites. Amongst these modification sites, one residue is localized in the juxtamembrane (Thr 654) and two are found in the catalytic domain (Ser 1046/1047) of the EGFR. We propose that, when EGFR is O-GlcNAc modified on Thr 654, EGFR may be transferred from early to late endosomes, whereas when EGFR is O-GlcNAc modified on Ser 1046/1047 desensitization of the receptor may be prevented. These findings suggest a complex interplay between phosphorylation and O-GlcNAc modification resulting in modulation of EGFR's functionality.     

The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor-insulin receptor generates Ca2+ signals through the PLC-gamma1 pathway.

The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

The ErbB4 extracellular region retains a tethered-like conformation in the absence of the tether

The epidermal growth factor receptor (EGFR) and its homologs ErbB3 and ErbB4 adopt a tethered conformation in the absence of ligand in which an extended hairpin loop from domain II contacts the juxtamembrane region of domain IV and tethers the domain I/II pair to the domain III/IV pair. By burying the hairpin loop, which is required for formation of active receptor dimers, the tether contact was thought to prevent constitutive activation of EGFR and its homologs. Amino‐acid substitutions at key sites within the tether contact region fail to result in constitutively active receptors however. We report here the 2.5 Å crystal structure of the N‐terminal three extracellular domains of ErbB4, which bind ligand but lack domain IV and thus the tether contact. This ErbB4 fragment nonetheless adopts a domain arrangement very similar to the arrangement adopted in the presence of the tether suggesting that regions in addition to the tether contribute to maintaining this conformation and inactivity in the absence of the tether contact. We suggest that the tether conformation may have evolved to prevent crosstalk between different EGFR homologs and thus allow diversification of EGFR and its homologs.     

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family—ErbB2, ErbB3, and ErbB4—contain a glycosylated extracellular ligand binding domain; a single-pass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3, 17, 36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in tethered, unliganded monomers to mediate dimer formation (7). Furthermore, an unusual asymmetric interface between two kinase domain monomers is linked to rearrangement of the kinase site to the active conformation (37). However, neither the position of the last extracellular domain, domain IV, nor association between the TM domains is well-defined experimentally in liganded receptors. The approximate location of domain IV has been suggested by models based on the orientation between domains III and IV in unliganded monomers (7, 12) and two-dimensional negative-stain electron microscopy (EM) averages (27); however, the position of domain IV in the liganded dimer has not been determined in previous crystal structures (13, 30). Thus, it is not known how the extracellular domain positions the TM domains for transmembrane signaling.Several lines of evidence suggest that the TM domain contributes directly to receptor dimerization and signaling. The neu oncogene encodes a Val → Glu substitution in the TM domain of ErbB2 that results in constitutive activation (34). Recombinant EGFR fragments consisting of the extracellular and TM domains have a 10⁵-fold higher affinity for dimerization than the isolated soluble extracellular domains (31). The TM domains of all four ErbB family members self-associate when expressed in bacterial inner membranes (26). A dimeric structure for isolated ErbB2 TM peptides in bicelles has been defined by nuclear magnetic resonance (NMR) imaging (4). However, ErbB2 does not bind ligand and does not physiologically homodimerize (17). Moreover, different ErbB family member TM domains utilize potentially distinct GxxxG sequence motifs to dimerize, as shown with fusion proteins in bacterial membranes (26). However, it is not clear how the TM domains contribute to dimerization and signaling in intact receptors on the cell surface.Here, we characterize the structural basis for EGFR transmembrane signaling. An improved crystal structure of the EGF-bound EGFR extracellular domain resolves domain IV in electron density maps and identifies a small domain IV dimerization interface, the mutation of which does not abolish signaling. The crystal structure and disulfide cross-linking demonstrate a flexible, dimeric linker between the extracellular and transmembrane domains. EGF-induced dimerization of the TM domains involves an interface far less extensive than that found in two receptors that dimerize in the absence of activation. Furthermore, mutagenesis shows that no unique interface is required for transmembrane signaling. Thus, we propose that signal transmission through the EGFR is communicated much more loosely than was previously thought.     

A dimerization hierarchy in the transmembrane domains of the HER receptor family

Bitopic membrane proteins offer an opportunity for studying transmembrane domain interactions without the structural complexity inherent to multitopic integral membrane proteins. To date, only homomeric associations have been extensively studied quantitatively. Here we propose to assess the thermodynamics of heteromeric associations, which opens the way to investigating specificity and selectivity. A very interesting system of biological relevance with single transmembrane domains possibly involved in interactions with different partners is the EGFR receptor family. The four members, all tyrosine kinase receptors, are involved in an interaction network that potentially leads to a complete set of homo- and heterodimers, ideally suited to such a study. Furthermore, the transmembrane domains of these receptors have been previously implicated in their function in the past by mutations in the transmembrane domain leading to constitutive activation. We demonstrate, using a fluorescence-based measurement of interaction energies, a hierarchy of transmembrane domain interactions ranging from a noninteractive pair to strong dimerization. We propose a structural model based on the crystal structure of the EGFR dimer, to show how the dimeric structure favors these interactions. The correlation we observe between transmembrane domain and whole receptor interaction hierarchies opens a new perspective, suggesting a role for transmembrane receptor domains in the modulation of receptor signaling.     

Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases

Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.     

Regulated migration of epidermal growth factor receptor from caveolae.

In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF. To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR. All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae. By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor. Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrin-coated pits. Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.     

Effect of carboxyl terminal truncation on the tyrosine kinase activity of the epidermal growth factor receptor.

The carboxyl terminal domain of the epidermal growth factor receptor (EGFR) is an important regulatory region in mediating the tyrosine kinase-dependent biological effects of EGF. The effect of a 164-amino-acid carboxyl deletion of the EGFR or the EGFR cytoplasmic kinase domain on in vitro tyrosine kinase activity was assessed. C'-terminal truncation of the EGFR resulted in dependence on Mn2+ for full activity. The EGFR kinase domain (kd EGFR) and the C'-terminally truncated kinase domain (kd c'1022 EGFR) also exhibited a strong preference for Mn2+ compared to Mg2+, with kd c'1022 EGFR being completely inactive in the presence of Mg2+ alone. Sphingosine or ammonium sulfate specifically activated both kd EGFR and kd c'1022 EGFR. EGFR and c'1022 EGFR displayed similar EGF-stimulated in vitro tyrosine kinase activities; however, kd EGFR was 5- to 10-fold more active in vitro than kd c'1022 EGFR. Thus, the regulatory contribution of the C'-terminus is most evident when the EGFR ligand binding domain is removed. These results indicate that an intact EGFR C'-terminus is necessary for the protein to assume a fully active conformation.     

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.     

Cell-free expression and purification of the fragments of the receptor tyrosine kinases of the EGFR family,containing the transmembrane domain with the juxtamembrane region,for structural studies

The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the ¹³C,¹⁵N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.     

NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.     

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.     

BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.     

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.     

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.