EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity.
ABSTRACT: We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279 , 38143-38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k(cat) approx. 5-fold, but does not significantly alter the K(m) for any ErbB peptides. Furthermore, peptides containing lysine at position -2 or -3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K(m) and increased the k(cat) for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.
Project description:The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2? (neuregulin 2?) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2?/Q43L may be an ErbB4 antagonist. NRG2?/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2?/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2?. In addition, NRG2? stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2? stimulates greater Akt phosphorylation than does NRG2?/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2? and NRG2?/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2?/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2?/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2?/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2?/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.
Project description:The human ErbB family of receptor tyrosine kinases comprises the epidermal growth factor receptor (EGFR/ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). ErbBs play fundamental roles in cell growth and differentiation events in embryonic and adult tissues, and inappropriate ErbB activity has been implicated in several human cancers. We report here the 2.4 A crystal structure of the extracellular region of human ErbB4 in the absence of ligand and show that it adopts a tethered conformation similar to inactive forms of ErbB1 and ErbB3. This structure completes the gallery of unliganded ErbB receptors and demonstrates that all human ligand-binding ErbBs adopt the autoinhibited conformation. We also show that the binding of neuregulin-1beta to ErbB4 and ErbB3 and the binding of betacellulin to both ErbB4 and ErbB1 does not decrease at low pH, unlike the binding of epidermal growth factor and transforming growth factor-alpha to ErbB1. These results indicate an important role for ligand in determining pH-dependent binding and may explain different responses observed when the same ErbB receptor is stimulated by different ligands.
Project description:ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.
Project description:Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1? that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions.
Project description:Neisseria meningitidis, the causative agent of meningitis and septicemia, attaches to and invades various cell types. Both steps induce and/or require tyrosine phosphorylation of host cell proteins. Here, we used a phospho array platform to identify active receptor tyrosine kinases (RTKs) and key signaling nodes in N. meningitidis-infected brain endothelial cells to decipher RTK-dependent signaling pathways necessary for bacterial uptake. We detected several activated RTKs, including the ErbB family receptors epidermal growth factor receptor (EGFR), ErbB2, and ErbB4. We found that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation and expression resulted in decreased bacterial uptake and heterologous expression of EGFR, ErbB2, or ErbB4 in Chinese ovary hamster (CHO-K1) cells, which do not express of EGFR and ErbB4; the decrease caused a significant increase in meningococcal invasion. Activation of EGFR and ErbB4 was mediated by transactivation via the common ligand HB-EGF (heparin-binding EGF-like ligand), which was significantly elevated in infected cell culture supernatants. We furthermore determined that N. meningitidis induced phosphorylation of EGFR at Tyr845 independent of ligand binding, which required c-Src activation and was involved in mediating uptake of N. meningitidis into eukaryotic cells. Increased uptake was repressed by expression of EGFR Y845F, which harbored a point mutation in the kinase domain. In addition, activation of ErbB4 at its autophosphorylation site, Tyr1284, and phosphorylation of ErbB2 Thr686 were observed. Altogether, our results provide evidence that EGFR, ErbB2, and ErbB4 are activated in response to N. meningitidis infection and shed new light on the role of ErbB signaling in meningococcal infection biology.
Project description:The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.
Project description:Interactions between short modified peptide motifs and modular protein domains are central events in cell signal-transduction. We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K. We identified STAT5 as a direct binding partner to EGFR and ErbB4 and discovered new recognition motifs for Shc and STAT5. The overall pattern of interaction partners of EGFR and ErbB4 suggests similar roles during signaling through their respective ligands. Phosphorylation kinetics of several tyrosine resides was measured by mass spectrometry and correlated with interaction partner preference. Our results demonstrate that system-wide mapping of peptide-protein interactions sites is possible, and suggest shared and unique roles of ErbB-receptor family members in downstream signaling.
Project description:As ErbB signaling is a determinant of prolactin synthesis, role of ErbB receptors was tested for prolactinoma outcomes and therapy. The objective of this study was to characterize ErbB receptor expression in prolactinomas and then perform a pilot study treating resistant prolactinomas with a targeted tyrosine kinase inhibitor (TKI). Retrospective analysis of prolactinomas and pilot study for dopamine agonist resistant prolactinomas in tertiary referral center. We performed immunofluorescent staining of a tissue array of 29 resected prolactinoma tissues for EGFR, ErbB2, ErbB3, and ErbB4 correlated with clinical features. Two patients with aggressive resistant prolactinomas enrolled and completed trial. They received lapatinib 1,250 mg daily for 6 months with tumor and hormone assessments. Main outcome measures were positive tumor staining of respective ErbB receptors, therapeutic reduction of prolactin levels and tumor shrinkage. Treated PRL levels and tumor volumes were suppressed in both subjects treated with TKI. EGFR expression was positive in 82 % of adenomas, ErbB2 in 92 %, ErbB3 in 25 %, and ErbB4 in 71 %, with ErbB2 score > EGFR > ErbB4 > ErbB3. Higher ErbB3 expression was associated with optic chiasm compression (p = 0.03), suprasellar extension (p = 0.04), and carotid artery encasement (p = 0.01). Higher DA response rates were observed in tumors with higher ErbB3 expression. Prolactinoma expression of specific ErbB receptors is associated with tumor invasion, symptoms, and response to dopamine agonists. Targeting ErbB receptors may be effective therapy in patients with resistant prolactinomas.
Project description:The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.
Project description:ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues-including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region--although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K(d) of approximately 1.1 microM. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened alphaC-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the "inactive-like" configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.