Interactome of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells

Abstract

Introduction: As a heterotrimeric enzyme, AMP-activated protein kinase (AMPK) is a major metabolic sensor in regulating cellular energy homeostasis, and is expressed on many tissues. In particular, AMPK contributes to insulin resistance associated with type 2 diabetes. Generally, cellular processes need tight control of protein kinases, which is influenced by through their formation of complex with other substrates. Despite their crucial function in regulation and pathogenesis, there are limited information on the interaction of protein kinases.

Methods: To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Prior to perform AP-MS, recombinant 6-myc tagged AMPKα1/β1 proteins were transiently transfected in INS-1. Immunoprecipitation was performed accompanied with two different beads: anti-myc antibody-conjugated agarose and magnetic beads. After in-gel trypsin digestion, peptides were analyzed in triplicate by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: Through a large scale analysis using AP-MS approach, we represented that our approach identified 381 unique proteins in the AMPKα1/β1 interactomes: 325 interactors of AMPK-α1 and 243 for AMPK-β1. The result from the magnetic beads capturing shows a higher number of identified interacting proteins than that from the agarose beads capturing. Furthermore, we identified 196 novel interactors that have not been previously characterized in the existing protein-protein interaction databases. Notably, our bioinformatics analysis suggests that the novel interactors mediated functions that are related to the regulation of actin cytoskeleton. Specifically, several proteins were linked to pancreatic beta-cell functions, including beta-cell development, beta-cell differentiation, glucose-stimulated insulin secretion, and cell-cell communication.

Conclusions: Our AMPK-specific interactome study suggests that our newly identified interactors could be valuable targets for the study of AMPK signaling pathways associated with pancreatic beta cell functions.

* This work is published in Scientific Reports Journal.