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

• Published in: Journal of proteomics Vol. 75; no. 11; pp. 3304 - 3313
• Main Authors: Klaus, Anna, Polge, Cécile, Zorman, Sarah, Auchli, Yolanda, Brunisholz, René, Schlattner, Uwe
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 18-06-2012; Elsevier
• Subjects: AMP-activated protein kinase; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Biochemistry, Molecular Biology; Biological and medical sciences; Cell physiology; cell signaling; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Diverse techniques; DNA Repair; energy; enzyme activity; fumarate hydratase; Fumarate Hydratase - genetics; Fumarate Hydratase - metabolism; Fundamental and applied biological sciences. Psychology; homeostasis; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Isoenzymes - genetics; Isoenzymes - metabolism; Life Sciences; liver; Molecular and cellular biology; noninsulin-dependent diabetes mellitus; phosphorylation; protein kinase substrates; protein phosphorylation; proteins; Rats; screening; Signal Transduction - physiology; Substrate Specificity - physiology; surface plasmon resonance; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; protein kinase substrates; protein phosphorylation; AMP-activated protein kinase; surface plasmon resonance; fumarate hydratase; cell signaling; Phosphorylation; Enzyme; Transferases; Non-specific serine/threonine protein kinase; Lyases; Activation; Identification; Surface plasmon; cAMP-dependent protein kinase; Protein; Cell signaling; Substrate; Fumarate hydratase; Resonance; Carbon-oxygen lyases; Hydro-lyases; Tumor suppressor gene; type ii diabetes; diabète de type 2; protéine kinase; résonance plasmonique de surface
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2012.03.040

AMP-activated protein kinase (AMPK) is emerging as a central cellular signaling hub involved in energy homeostasis and proliferation. The kinase is considered as a suitable target for pharmacological intervention in several energy-related pathologies like diabetes type II and cancer, although its signaling network is still incompletely understood. Here we apply an original two-dimensional in vitro screening approach for AMPK substrates that combines biophysical interaction based on surface plasmon resonance with in vitro phosphorylation. By enriching for proteins that interact with a specific AMPK isoform, we aimed to identify substrates that are also preferentially phosphorylated by this specific AMPK isoform. Application of this screen to full-length AMPK α2β2γ1 and soluble rat liver proteins identified the tumor suppressor fumarate hydratase (FH). FH was confirmed to interact with and to be preferentially phosphorylated by the AMPKα2 isoform by using yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of FH significantly increased enzyme activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. In vivo, AMPKα2 is supposed to target the cytosolic/nuclear pools of FH, whose tumor suppressor function relies on DNA damage repair and inhibition of HIF-1α-signaling.
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► In vitro screening protocol for protein kinase substrates. ► Combining interaction (surface plasmon resonance) and phosphorylation assays. ► Tumor suppressor fumarate hydratase (FH) is a preferential AMPK- α2 substrate. ► FH phosphorylation leads to enzymatic activation.