Asymmetric kinase dimer formation is crucial for the activation of oncogenic EGFRvIII but not for ERBB3 phosphorylation
© Kancha et al.; licensee BioMed Central Ltd. 2013
Received: 11 November 2012
Accepted: 27 May 2013
Published: 10 June 2013
Formation of asymmetric kinase dimers is required for wt-EGFR activation upon ligand stimulation. The role of receptor dimerization in oncogenic EGFRvIII mutant activation is not completely understood and the molecular details of EGFRvIII interactions within homo-dimers and hetero-dimers are not elucidated yet.
By employing mutations that disrupt the asymmetric kinase dimer interface in EGFRvIII, we demonstrate that the mechanism of oncogenic EGFRvIII mutant activation is similar to that of the full-length wild-type EGFR. Surprisingly, the monomeric EGFRvIII lacks autophosphorylation and the formation of asymmetric kinase dimers is indispensable for oncogenic kinase activation. In addition, we show that ERBB3 can act as an activator of EGFRvIII by forming asymmetric kinase dimer in a ligand-independent manner. Interestingly, we found that the formation of asymmetric kinase dimer is dispensable for ERBB3 phosphorylation by the activated EGFR kinase as well as the ERBB2 kinase thus revealing a novel model for receptor function.
Lateral signaling is a novel mechanism of signal propagation via ERBB3 upon activation by EGFR/ERBB2 kinase even in the absence of their ability to form asymmetric kinase dimers.
EGFRvIII monomers lack kinase activity
To further demonstrate the importance of asymmetric dimer formation for kinase activity, cells were transfected with a combination of mutants wherein the kinase activity of a C-lobe mutant (V948R) is rescued in trans by the kinase-dead (D837N) EGFRvIII either alone (Figure 1A-B, lane 7) or in combination with the N-lobe mutant (Figure 1A-B, lane 10). In contrast, the activity of C-lobe (V948R) mutant could not be rescued by a kinase-dead C-lobe mutant (D837N + V948R) due to the disruption of the asymmetric kinase dimer interface (Figure 1A-B, lane 13). Additional EGFRvIII mutants with disrupted asymmetric kinase dimer interface both in wild-type and D837N background were taken as controls (lanes 6, 8, 9, 11 and 12) to demonstrate the absence of cis-autophosphorylation (Figure 1A-B, Additional file 2: Figure S1A). Together these data argue for an important role of asymmetric dimer formation also for EGFRvIII kinase activation. Furthermore, it shows for the first time that the extra-cellular in-frame deletion of the EGFRvIII receptor does not result in an activated monomer as previously anticipated. A recent study reported the importance of Cys307 (wild-type EGFR numbering; Cys16 in mature EGFRvIII mutant) in EGFRvIII receptor dimerization . We thus cloned the C16S mutant into the EGFRvIII/D837N backbone and tested for its ability to activate the C-lobe mutated EGFRvIII. As expected, the EGFRvIII/C16S + D837N mutant was not able to activate the EGFRvIII/V948R mutant indicating that the receptor dimerization is indispensable for EGFRvIII activity (Additional file 2: Figure S1B).
ERBB3 is an activator of EGFRvIII in an asymmetric kinase hetero-dimer
Recently, it was shown that ERBB3 could act as an activator for the wt-EGFR kinase . However, it is not known whether oncogenic EGFRvIII is able to form activating dimers with ERBB3. To test for potential ERBB3/EGFRvIII interactions, we expressed both constructs in HEK293 cells, which lack ERBB receptor expression [8, 12] (Figure 1C: lanes 1–2, Additional file 3: Figure S2A). ERBB3 lacks intrinsic kinase activity  and when expressed alone didn’t cause receptor phosphorylation even in the presence of it’s ligand heregulin (Figure 1C: lanes 5–6, Additional file 3: Figure S2A). However, expression of ERBB3 together with EGFRvIII mutant resulted in ERBB3 phosporylation indicating that ERBB3 can act as a substrate for EGFRvIII kinase by forming heterodimers (Figure 1C-D: lanes 7–8). Interestingly, EGFRvIII-I706Q (N-lobe) mutant that disrupts asymmetric kinase dimer formation didn’t result in ERBB3 phosphorylation indicating that ERBB3 acts as an activator of EGFRvIII kinase (Figure 1C: lanes 9–10, Additional file 3: Figure S2A). Moreover, ERBB3 rescued the kinase activity of EGFRvIII-V948R (C-lobe) mutant thus demonstrating that the asymmetric kinase dimer interface is similar for both the EGFRvIII homodimers and EGFRvIII-ERBB3 heterodimers (Figure 1C-D: lanes 11–12). Receptor phosphorylation also correlated with the phosphorylation of downstream targets such as STAT5 indicating that the asymmetric kinase dimers are indeed functional. The phenomenon of EGFR kinase activation by asymmetric kinase dimerization thus seems to be highly conserved among different members of the EGFR family and different types of activating mutations found in human cancers [7, 13]. Since kinase-inactive EGFRvIII is still able to be an activator for a partner receptor, inhibitor-bound-EGFRvIII may still activate other receptors of the EGFR family (ERBB2 or ERBB4). In the setting of EGFR tyrosine kinase inhibitor (TKI) treatment this may lead to altered signal transduction and secondary drug resistance. Thus, the expression of ERBB2, ERBB3 and ERBB4 in EGFR driven cancer may be important to predict the outcome of TKI treatment.
Asymmetric kinase dimerization is dispensible for ERBB3 phosphorylation by activated EGFR and ERBB2 kinases
Activated ERBB3 potentiates the transforming ability of EGFR and ERBB2
Formation of asymmetric kinase dimer is essential for both the constitutive activation of oncogenic EGFR as well as the ligand-stimulated wild-type EGFR. However, phosphorylation of ERBB3 by the activated EGFR or ERBB2 kinase may occur in higher order oligomers in the absence of asymmetric kinase dimer formation. Thus, asymmetric kinase dimer formation plays a differential role in EGFR receptor activation and ERBB3 phosphorylation. Recent studies have implicated the role of ERBB3 as a critical heterodimeric partner for both EGFR and ERBB2 in drug resistance . Since the formation of receptor complexes is important for their activity, the use of antibodies that target ERBB receptors either alone or in combination with ERBB inhibitors might abrogate the development of secondary drug resistance.
Justus Duyster is supported by a grant from the José Carreras Leukämie-Stiftung. The authors thank Natalie Bartosch for technical support.
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