Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha 1.
ABSTRACT 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: The combination of an increase in the cAMP-phosphodiesterase activity of h-prune and its interaction with nm23-H1 have been shown to be key steps in the induction of cellular motility in breast cancer cells. Here we present the molecular mechanisms of this interaction. The region of the nm23-h-prune interaction lies between S120 and S125 of nm23, where missense mutants show impaired binding; this region has been highly conserved throughout evolution, and can undergo serine phosphorylation by casein kinase I. Thus, the casein kinase I delta-epsilon specific inhibitor IC261 impairs the formation of the nm23-h-prune complex, which translates 'in vitro' into inhibition of cellular motility in a breast cancer cellular model. A competitive permeable peptide containing the region for phosphorylation by casein kinase I impairs cellular motility to the same extent as IC261. The identification of these two modes of inhibition of formation of the nm23-H1-h-prune protein complex pave the way toward new challenges, including translational studies using IC261 or this competitive peptide 'in vivo' to inhibit cellular motility induced by nm23-H1-h-prune complex formation during progression of breast cancer.Oncogene 04/2008; 27(13):1853-64. DOI:10.1038/sj.onc.1210822 · 8.56 Impact Factor
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ABSTRACT: The Skp-Cul-F box (SCF) ubiquitin E3 ligase machinery recognizes predominantly phosphodegrons or, less commonly, an I/LQ molecular signature within substrates to facilitate their recruitment in mediating protein ubiquitination and degradation. Here we examined the molecular signals that determine the turnover of the multifunctional enzyme nucleoside diphosphate kinase A (NDPK-A) that controls cell proliferation. NDPK-A protein exhibits a half-life of ∼6 h in HeLa cells and is targeted for ubiquitylation through actions of the F-box protein FBXO24. SCF-FBXO24 polyubiquitinates NDPK-A at K85 and two NH2-terminal residues, L55 and K56, were identified as important molecular sites for FBXO24 interaction. Importantly, K56 acetylation impairs its interaction with FBXO24 and substitution of K56 with Q56, an acetylation mimic, reduces NDPK-A FBXO24 binding capacity. The acetyltransferase GCN5 catalyzes K56 acetylation within NDPK-A thereby stabilizing NDPK-A, whereas GCN5 depletion in cells accelerates NDPK-A degradation. Cellular expression of a NDPK-A acetylation mimic or FBXO24 silencing increases NDPK-A lifespan that in turn impairs cell migration and wound healing. We propose that lysine acetylation when presented in the appropriate context may be recognized by some F box proteins as a unique inhibitory molecular signal for their recruitment to restrict substrate degradation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Molecular and Cellular Biology 01/2015; 35(6). DOI:10.1128/MCB.01185-14 · 5.04 Impact Factor
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ABSTRACT: Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.Journal of Biological Chemistry 10/2008; 283(36):25103. DOI:10.1074/jbc.M610956200 · 4.60 Impact Factor