EYA1's conformation-specificity in dephosphorylating phosphothreonine in Myc and its activity on Myc stabilization in breast cancer

Abstract

EYA1 is known to be overexpressed in human breast cancer in which Myc protein is also accumulated in association with decreased phospho-T58 levels. We have recently reported that EYA1 functions as a unique protein phosphatase to dephosphorylate Myc at phosphor-T58 to regulate Myc levels. However, it remains unclear whether EYA1-mediated Myc dephosphorylation on T58 1 is a critical function in regulating Myc protein stability in breast cancer. Furthermore, EYA1's substrate specificity has remained elusive. In this study, we have investigated these questions and here we report that depletion of EYA1 using shRNA in breast cancer cells destabilizes Myc protein and increases pT58 levels, leading to an increase in doubling time and impairment of cell cycle progression. In correlation with EYA1-mediated stabilization of cMyc and reduced levels of pT58, EYA1 greatly reduced cMyc-FBW7 binding and cMyc ubiquitination, thus providing novel insight into how EYA1 acts to regulate the FBW7-mediated Myc degradation machinery. We find that the conserved C-terminal haloacid dehalogenase domain of EYA1, which has been reported to only have tyrosine-phosphatase activity, has dual phosphatase activity and both the N- and C-terminal domains interact with substrates to increase catalytic activity of EYA1. Enzymatic assay and NMR (nuclear magnetic resonance) analysis demonstrate that EYA1 has striking conformation preference toward phospho-T58 of Myc. Together, our results not only provide novel structural evidence on conformation-specificity of EYA1 in dephosphorylating phosphothreonine in Myc but also reveal an important mechanism contributing to Myc deregulation in human breast cancer.