Understanding the Molecular Basis of the Interaction between NDPK-A and AMPK α1
Department of Maternal and Child Health Sciences, University of Dundee, Dundee DD1 9SY, United Kingdom.Molecular and Cellular Biology (Impact Factor: 4.78). 09/2006; 26(15):5921-31. DOI: 10.1128/MCB.00315-06
Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 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 alpha1 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 alpha1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK alpha1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK alpha1 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 alpha1 binding and substrate channeling.
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ABSTRACT: Previously we elucidated the molecular interaction between the nucleoside diphosphate kinase A (NDPK-A)/AMP-activated protein kinase (AMPK) alpha1 complex, discovering a process we termed "substrate channeling." Here, we investigate the protein-protein interaction of the substrate channeling complex with the pleiotropic protein kinase, CK2 (formerly casein kinase 2). We show that CK2 is part of the NDPK-A/AMPK alpha1 complex under basal (background AMPK activity) conditions, binding directly to each of the complex components independently. We report that when S122 on NDPK-A is phosphorylated by AMPK alpha1 in vivo, (i.e., stimulation of AMPK using either metformin or phenformin) initiating the substrate channeling mechanism, the catalytic subunit of CK2 (CK2alpha) is expelled from the complex and translocates to bind NDPK-B, a closely related but independent isoform of NDPK. Thus, we find that the AMPK-dependent phospho-status of S122 on NDPK-A determines whether CK2alpha swaps partners between NDPK-A and NDPK-B. This is the first reported linkage between NDPK-A and NDPK-B via a phosphorylation pathway and could explain the complex biology of NDPK. This study also offers an explanation as to how CK2alpha exclusion mutations (S120A or S122D of NDPK-A) on NDPK-A might have implications in cancer biology and general cellular energy metabolism.The FASEB Journal 02/2007; 21(1):88-98. DOI:10.1096/fj.06-6804com · 5.04 Impact Factor
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ABSTRACT: It is generally accepted that G protein coupled receptors (GPCR) activate heterotrimeric G proteins by inducing a GDP/GTP exchange at the G protein alpha subunit. In addition, the transfer of high energetic phosphate by nucleoside diphosphate kinase (NDPK) and/or the beta subunit of G proteins (Gbeta) can induce G protein activation. Recent evidence suggests that the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers. In this complex, NDPK B acts as a protein histidine kinase phosphorylating Gbeta at histidine residue 266 (His266). The high energetic phosphoamidate bond on His266 allows for a phosphate transfer specifically onto GDP and thus local formation of GTP, which binds to and thereby activates the respective G protein alpha subunit. Apparently, this process occurs independent of the classical GPCR-induced GDP/GTP exchange at least for members of the G(s) and G(i) subfamilies of heterotrimeric G proteins. By using a mutant of Gbeta(1) in which His266 was replaced by Leu, it was recently demonstrated that NDPK B/Gbetagamma-mediated G(s) activation contributes by about 50% to basal cAMP formation and contractility in rat cardiac myocytes. Besides its apparent role in G protein activation, the complex formation of NDPK B with Gbetagamma dimers might be essential for G protein stability. Depletion of either the NDPK B orthologue or Gbeta(1) isoforms in zebrafish embryos led to a similar phenotype displaying contractile dysfunction in the heart accompanied by a complete loss of heterotrimeric G protein expression. In conclusion, the interaction of NDKP B with Gbetagamma dimers might play an important role in signal transduction, and alterations in this novel pathway might be of pathophysiological importance.Archiv für Experimentelle Pathologie und Pharmakologie 03/2007; 374(5-6):373-83. DOI:10.1007/s00210-006-0126-6 · 2.47 Impact Factor
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ABSTRACT: Nucleoside diphosphate kinase A (NDPK-A) regulates the alpha1 isoform of the AMP-activated protein kinase (AMPK alpha1) selectively, independent of [AMP] and surrounding [ATP], by a process termed substrate channelling. Here, we show, using a range of empirically validated biochemical techniques, that the muscle form (M-LDH or LDH-A) and the heart form (H-LDH or LDH-B) of lactate dehydrogenase are physically associated with the liver cytosolic substrate-channelling complex such that M-LDH associates with NDPK-A, AMPK alpha1 and casein kinase 2 (CK2), whereas H-LDH associates with local NDPK-B. We find that the species of LDH bound to the substrate-channelling complex regulates the in vivo enzymatic activities of both AMPK and CK2, and has a downstream effect on the phospho-status of acetyl CoA carboxylase, a key regulator of cellular fat metabolism known to be a part of the cytosolic substrate-channelling complex in vivo. We hypothesise that the regulatory presence of LDH in the complex couples the substrate-channelling mechanism to both the glycolytic and redox states of the cell, allowing for efficient sensing of cell metabolic status, interfacing with the substrate-channelling complex and regulating the enzymatic activities of AMPK and CK2, two critical protein kinases.Journal of Molecular Biology 09/2007; 371(2):349-61. DOI:10.1016/j.jmb.2007.05.081 · 4.33 Impact Factor
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