Abstract
This study describes a novel in vitro two-dimensional screening approach for kinase substrates that combines a biophysical interaction assay based on surface plasmon resonance and a standard in vitro phosphorylation assay. The interaction assay was optimized with appropriate prepurification procedures on a Biacore instrument. It selects for substrates interacting with a specific kinase, and can thus identify substrates that are preferentially phosphorylated e.g. by a specific kinase isoform. This approach was applied to isoforms of the heterotrimeric AMP-activated protein kinase (AMPK). AMPK is an emerging central cellular signaling hub in energy homeostasis and proliferation, but its signaling network is still incompletely understood. Using soluble rat liver proteins and full-length AMPK α2-β2-γ1 complex, several putative AMPK substrates were identified by mass spectrometry. One of them, fumarate hydratase (fumarase), was confirmed as an in vitro AMPK target which preferentially interacted with and was phosphorylated by the AMPKα2 isoform as shown by yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of fumarate hydratase led to significant activation of enzymatic activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. This may have different physiological consequences, since the enzyme has a dual localization in the mitochondrial matrix and the cytosol. [AK and CP contributed equally to this work]
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call