Cyclictrans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

Abstract

Despite intensive research no therapies targeted against the oncogenic EGFRvIII are present in the clinic. One of the reasons is the elusive nature of the molecular structure and activity of the truncated receptor. The recent publications indicate the EGF-bound wild-type EGFR to trans-phosphorylate the EGFRvIII initiating aberrant signaling cascade. The elevated stability of the mutant receptor contributes towards oncogenic potential, preventing termination of signaling by receptor degradation. Here, we show that inhibition of phosphatases leads to a marked increase in phosphorylation of wild-type EGFR and EGFRvIII, indicating that both undergo cyclic rounds of phosphorylation and dephosphorylation on all investigated tyrosine residues, including Tyr1045. Still, we observe elevated stability of the mutant receptor, suggesting phosphorylation as insufficient to cause degradation. Hyperphosphorylation of EGFRvIII was hindered only by EGFR tyrosine kinase inhibitors. Co-immunoprecipitation as well as semi-native Western blotting structural analyses together with functional investigation of EGFRvIII’s phosphorylation following depletion of wild-type EGFR by shRNA or EGF-mediated degradation indicated homodimerization as the predominant quaternary structure of the mutant receptor. Dimers were observed only under non-reducing conditions, suggesting that homodimerization is mediated by covalent bonds. Previous reports indicated cysteine at position 16 to mediate covalent homodimerization. Upon its substitution to serine, we have observed impaired formation of dimers and lower phosphorylation levels of the mutated oncogene. Based on the obtained results we propose that EGFRvIII is predominantly regulated dynamically by phosphatases that counteract the process of trans-phosphorylation occurring within the homodimers.

EGFRvIII: An Oncogene with Ambiguous Role

“…Importantly, it has to be emphasized that EGFR vIII exhibits constitutive activity [121,122]. Our team demonstrated that mutant phosphorylation is elevated when compared to nonstimulated EGFR WT , while data obtained by other research teams indicate that constitutive EGFR vIII signaling corresponds to low level of signal intensity induced by ligand-activated EGFR WT [17,[123][124][125]. Such data indicate that EGFR vIII dimer conformation resembles inactive dimers of EGFR WT , thus suggesting that impact of this oncogenic receptor on cell biology is not a result of some specific, dimerization-related kinase activity, but rather a consequence of unique membrane stability [121,122].…”

“…Additionally, our data indicate that EGFR vIII signaling may not be associated with slightly elevated kinase activity, but rather its minimally lower sensitivity to phosphatase activity, when compared to wild-type receptor [123]. Moreover, we indicated that enhanced phosphorylation of tyrosine 1045 did not result in EGFR vIII degradation [123], suggesting that the previous model of impaired EGFR vIII degradation requires an update [121]. Nevertheless, without membrane stability, EGFR vIII signaling will not be strong enough to induce a biological effect.…”

“…Despite the fact that EGFR vIII stability seems to be more important than its kinase activity, the latter feature is still required to fully exhibit the oncogenic potential of this receptor. Such potential is dependent on signal transduction induced by phosphorylated tyrosines (at least in a model explaining EGFR vIII oncogenicity as a membrane receptor), especially as EGFR vIII was demonstrated to undergo constant phosphorylation and dephosphorylation cycles [123]. Additionally, our data indicate that EGFR vIII signaling may not be associated with slightly elevated kinase activity, but rather its minimally lower sensitivity to phosphatase activity, when compared to wild-type receptor [123].…”

“…Such data indicate that EGFR vIII dimer conformation resembles inactive dimers of EGFR WT , thus suggesting that impact of this oncogenic receptor on cell biology is not a result of some specific, dimerization-related kinase activity, but rather a consequence of unique membrane stability [121,122]. erefore, constitutive activity of EGFR vIII is not particularly high, but when combined with high membrane stability, it may enable triggering of some significant biological effects by this oncogenic receptor [17,[123][124][125].…”

“…Such potential is dependent on signal transduction induced by phosphorylated tyrosines (at least in a model explaining EGFR vIII oncogenicity as a membrane receptor), especially as EGFR vIII was demonstrated to undergo constant phosphorylation and dephosphorylation cycles [123]. Additionally, our data indicate that EGFR vIII signaling may not be associated with slightly elevated kinase activity, but rather its minimally lower sensitivity to phosphatase activity, when compared to wild-type receptor [123]. Moreover, we indicated that enhanced phosphorylation of tyrosine 1045 did not result in EGFR vIII degradation [123], suggesting that the previous model of impaired EGFR vIII degradation requires an update [121].…”