Abstract
The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.