Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation
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Wojciech Stec1,#, Kamila Rosiak1,2,#, Cezary Treda1,2, Maciej Smolarz1, Joanna Peciak1,2, Marcin Pacholczyk1,3, Anna Lenart1, Dawid Grzela1, Ewelina Stoczynska-Fidelus1,2,* and Piotr Rieske1,2,4,*
1Research and Development Unit, Celther Polska Ltd., Lodz, Poland
2Department of Tumor Biology, Medical University of Lodz, Lodz, Poland
3Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland
4Research and Development Unit, Personather Ltd., Lodz, Poland
#Contributed equally as first authors
*Contributed equally as senior authors
Piotr Rieske, email: firstname.lastname@example.org
Keywords: EGFR; EGFRvIII; dimerization; phosphorylation
Received: January 17, 2017 Accepted: December 29, 2017 Published: January 06, 2018
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.
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