Understanding the Molecular Basis of the Interaction between NDPK-A and AMPK α1

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (similar to 16 to 20 kDa) including two well-characterized isoforms (NDPK-A and -B). NDPK catalyzes the conversion of nucleoside diphosphates to nucleoside triphosphates, regulates a diverse array of cellular events, and can act as a protein histidine kinase. AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that responds to the cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of acetyl coenzyme A carboxylase 1 (ACC1), a regulator of cellular fatty acid synthesis. We recently reported that NDPK-A (but not NDPK-B) selectively regulates the alpha 1 isoform of AMPK independently of the AMP concentration such that the manipulation of NDPK-A nucleotide trans-phosphorylation activity to generate ATP enhanced the activity of AMPK. This regulation occurred irrespective of the surrounding ATP concentration, suggesting that "substrate channeling" was occurring with the shielding of NDPK-generated ATP from the surrounding medium. We speculated that AMPK alpha 1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK alpha 1 in vivo. We find that NDPK-A 5122 and 5144 are phosphorylated by AMPK alpha 1 and that the phosphorylation status of 5122, but not 5144, determines whether substrate channeling can occur. We report the cellular effects of the S122 mutation on ACC1 phosphorylation and demonstrate that the presence of E124 (absent in NDPK-B) is necessary and sufficient to permit both AMPK alpha 1 binding and substrate channeling.