ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

Abstract

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) residuesincluding the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular regionalthough 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 μM. 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 αC-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.dimerization | kinase inhibitor | catalytic mechanism | activation loop R eceptor tyrosine kinases (RTKs) from the EGF receptor (EGFR) or ErbB/HER family play important roles in animal development and disease (1) and are the targets of several important therapeutic agents used clinically to treat cancer. Each receptor contains a large extracellular ligand-binding region (targeted by therapeutic antibodies), a single transmembrane helix, and an intracellular tyrosine kinase domain (TKD) that is flanked by juxtamembrane and C-terminal regulatory regions and is targeted by specific small-molecule kinase inhibitors (1, 2). Ligand binding to the extracellular region promotes homo-or heterodimerization of ErbB receptors, leading to allosteric activation of their intracellular kinase domains through the formation of asymmetric dimers (3-5). Within an activated dimer, the C-terminal regulatory tail is trans-autophosphorylated on tyrosines and recruits downstream signaling molecules that contain phosphotyrosine-binding Src homology-2 (SH2) domains.ErbB3/HER3 is unique among the mammalian ErbB receptors in being generally considered as kinase-inactive (6). When first cloned (7,8), amino acid substitutions were noted at two particular sites that are conserved in other known kinases (9). ...

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…The ErbB2 protein lacks a ligand-binding domain and relies on heterodimerisation for signalling functionality (though may independently exert cardiac effects when abnormally expressed). ErbB3 lacks the ability to phosphorylate exogenous peptides, heterodimerising with ErbB receptors, in particular ErbB2 (Shi et al, 2010;Steinkamp et al, 2014) to confer a unique pro-survival properties, especially relating to tumor progression .…”

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“…ErbB3 kinase also follows a direct phosphorylation mechanism where the substrate (tyrosine) acts as a proton donor, donating a proton onto the γ-phosphate during phosphorylation. 42 Ting Shi et al disclosed a catalytic acid mechanism for CheA histidine kinase where the β-, γ-bridging oxygen of ATP forms a hydrogen-bond network with two water molecules, which participate in the protein relay from the catalytic acid Lys48 to the β-, γ-bridging oxygen of the ATP. 43 A catalytic base mechanism was suggested for cyclin-dependent protein kinases, where Asp127 acts as a catalytic base to abstract a proton from the substrate serine, which attacks the γ-phosphate of ATP.…”

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“…48,49 Initially, HER3, unlike EGFR and HER2, has been considered an unsuitable target for cancer treatments. This was due to an overall moderate expression levels in cancer cells, a catalytically impaired kinase domain, 50 a presumed lack of ability to form homodimeric HER3-HER3 complexes, and an initial inability to detect oncogenic mutations of HER3/ERBB3. Yet, it has been clear early on that HER3-containing heterodimers, especially with HER2, generate strong survival signals.…”

“…50 In addition, HER3 seems to act as an auxiliary subunit of driver oncogenes, such as HER2/ERBB2 and EGFR, rather than a bona fide driver. Yet another feature, which is less understood, is the ability of HER3 to instigate compensatory feedback regulatory loops that adapt and compensate for inhibition of other receptors of the HER/ERBB family.…”