Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates

Cell Signal. 2019 May;57:45-57. doi: 10.1016/j.cellsig.2019.02.001. Epub 2019 Feb 14.

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.

Keywords: GTPase activating protein and VPS9 domains 1; Shokat; Starch-binding domain 1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Animals
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Hepatocytes / metabolism
  • Homeostasis / genetics
  • Homeostasis / physiology
  • Lipid Metabolism / genetics
  • Liver / metabolism*
  • Mass Spectrometry / methods
  • Membrane Proteins / metabolism*
  • Mice, Knockout
  • Muscle Proteins / metabolism*
  • Phosphorylation
  • Substrate Specificity

Substances

  • Gapex-5 protein, mouse
  • Guanine Nucleotide Exchange Factors
  • Membrane Proteins
  • Muscle Proteins
  • starch-binding domain-containing protein 1, mouse
  • AMP-Activated Protein Kinases