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

Abstract

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (ϳ16 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 ␣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 ␣1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK ␣1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK ␣1 and that the phosphorylation status of S122, but not S144, 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 ␣1 binding and substrate channeling.Nucleoside diphosphate kinase (NDPK) (nm23/awd) has been recognized for over 40 years as a ubiquitous enzyme that catalyzes the transfer of the ␥-phosphate of a nucleoside triphosphate to a different nucleoside diphosphate (14). For example, the addition of ADP to GTP generates ATP plus GDP. This versatility allows NDPK to function as a nucleotide converter, balancing different cytosolic nucleotide pools, but cannot explain the necessity for nine human isoforms (Nm23-H1 to Nm23-H9) transcribed from different nm23 genes, constituting a family of structurally and functionally conserved proteins with differing functions (23). NDPK was also reported to be a protein histidine kinase (9), and the most widely expressed isoforms of NDPK (NDPK-A and -B) regulate a diverse array of cellular events including growth and development, tumor metastasis, and transcriptional regulation (9, 25). Despite sharing 88% sequence identity, these two isoforms have distinct cellular functions (3), and accumulating evidence indicates that protein-protein interactions can modulate the specific molecular actions of NDPK, with new binding partners being identified at an increasing rate (25). One such example is our recent report that an NDPK-A/AMP-activated protein kinase (AMPK) ␣1 complex associates with the cystic fibrosis transme...

M-LDH Serves as a Regulatory Subunit of the Cytosolic Substrate-channelling Complex in Vivo

“…array approach that has recently been validated empirically. 19,24 We found that both M-LDH and H-LDH exhibit the same in vitro binding capabilities, binding the AMPK α1, AMPK β1, AMPK β2, AMPK γ1 and AMPK γ2 directly, but not the AMPK α2 isoforms of AMPK, an observation also made in the in vivo precipitation in Figure 1(b). Interestingly, our results also show that both LDH isoforms are equally capable of binding CK2β, NDPK-A and NDPK-B, but not CK2α.…”

“…19,24 The four peptides (NDPKpep 1, 2, 3 and 4) correspond to exposed regions of NDPK-A (amino acid residues 49-62, 92-105, 111-125 and 139-152, respectively), as determined by crystal structure with peptide 3 forming the interaction/binding region of NDPK-A for both AMPK α1 and CK2α. Figure 3(b) shows that M-LDH and H-LDH exhibit the same binding capabilities, interacting directly with/binding the AMPK α1, AMPK β1, AMPK β2, AMPK γ1 and AMPK γ2, but not the AMPK α2 isoform, similar to our finding shown in Figure 1(b), where LDH does not interact with AMPK α2 in vivo.…”

Protein Kinase CK2, Cystic Fibrosis Transmembrane Conductance Regulator, and the ΔF508 Mutation

“…The CF antigen, which is overexpressed in both CF patients and model systems, is a regulator of CK2 kinase activity (48,49). Finally, our earlier studies demonstrated defective function of NDPK-A in CF cells (50), and NDPK is an in vivo CK2 target (51) that we have recently shown to not only regulate AMPK ␣1 but also to bind to CFTR (21,52). Thus, some of the reported targets of CK2 (27,53) include proteins controlling unexplained aspects of CF disease (50,54).…”

“…Immunoblotting-Immunoblotting was carried out as described previously (21). Briefly, blotted membranes were blocked for 30 min in TBS/Tween with 5% milk powder; and anti-NBD1 antibody (Neomarkers) was applied for 1.5 h followed by four 15-min washes, and then horseradish peroxidase-labeled anti-mouse secondary antibody was applied at a 1:5000 dilution for 45 min followed by four 15-min washes.…”

The Ubiquitin E3 Ligase SCF-FBXO24 Recognizes Deacetylated Nucleoside Diphosphate Kinase A To Enhance Its Degradation

“…For example, NDPK-A activity is stringently regulated by 5= AMP-activated protein kinase (AMPK)-mediated phosphorylation, where AMPK␣1 interacts with NDPK-A via E124 to phosphorylate two adjacent residues, S122 and S144 (24)(25)(26). AMPK phosphorylates NDPK-A at S122 that augments its enzymatic activity by triggering substrate channeling.…”