A two-dimensional screen for AMPK substrates identifies tumor suppressor fumarate hydratase as a preferential AMPKα2...

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Signaling

Background:

• AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and cellular proliferation.
• Understanding the complete AMPK signaling network is crucial for developing treatments for metabolic diseases like type II diabetes and cancer.
• Pharmacological targeting of AMPK holds promise, but requires a deeper knowledge of its substrates and regulatory mechanisms.

Purpose of the Study:

• To identify novel substrates of AMP-activated protein kinase (AMPK), specifically those phosphorylated by the AMPKα2 isoform.
• To investigate the functional consequences of AMPK-mediated phosphorylation on identified substrates.
• To elucidate the role of AMPK-regulated fumarate hydratase (FH) in cellular processes and disease.

Main Methods:

• A novel two-dimensional in vitro screening approach combining surface plasmon resonance (SPR) for interaction analysis and in vitro phosphorylation assays.
• Enrichment of proteins interacting with a specific AMPK isoform (AMPKα2β2γ1) to identify preferential substrates.
• Validation of substrate identification using yeast-two-hybrid assays and in vitro phosphorylation experiments.
• Assessment of enzyme activity changes upon phosphorylation in vitro and in vivo.

Main Results:

• The screening identified fumarate hydratase (FH), a known tumor suppressor, as a substrate of the AMPKα2 isoform.
• FH was confirmed to interact with and be preferentially phosphorylated by AMPKα2.
• AMPK-mediated phosphorylation significantly increased FH enzyme activity both in vitro and in vivo.
• Evidence suggests AMPKα2 targets cytosolic and nuclear FH, impacting DNA repair and HIF-1α signaling.

Conclusions:

• Fumarate hydratase (FH) is a direct substrate of AMP-activated protein kinase (AMPK), specifically the AMPKα2 isoform.
• Phosphorylation by AMPK enhances FH enzyme activity, suggesting a regulatory role in cellular metabolism and tumor suppression.
• This finding provides a mechanistic link between energy sensing by AMPK and the tumor suppressor functions of FH, relevant to cancer and metabolic disorders.