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Query: EC:2.7.11.31 (AMP-activated protein kinase)
 13,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity. One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments. AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic [32P]phosphopeptide containing exclusively phospho-Ser621. AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.
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PMID:Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase. 909 12

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.
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PMID:Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. 1086 86

We have expressed a truncated form of the alpha1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/alpha (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P-16 to P-5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P-6 and P-4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P+3 interacts with D56 on the small lobe; (iv) the side-chain of P+4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the gamma-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.
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PMID:Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate. 1190 45

SNARK, the fourth member of the AMPK catalytic subunit family, was originally identified in a rat kidney cDNA library, and in this study we isolated its human homologue. A BLAST search analysis using rat SNARK protein yielded a single high homology clone, DKFZp434J037, isolated from human testis, and since its hypothetical protein showed 84% homology to rat SNARK protein, we assumed DKFZp434J037 to be the human SNARK cDNA. The human SNARK cDNA is 3443bp long and encodes a 628 amino acid protein having an estimated molecular weight of 69kDa, and its chromosomal localization had been assigned to 1q32.1. The same as other members of AMPK catalytic subunit family, human SNARK showed AMP-dependent GST-SAMS phosphorylation activity and enhanced HepG2 cell survival during glucose starvation. Human SNARK-overexpressing HepG2 cells (H/SNK) showed acute cell-cell detachment when exposed to glucose-free medium and the cell-cell detachment correlated well with the detection of G-actin. Deletion mutant analysis strongly suggested that the putative catalytic domain of SNARK is necessary for the cell-cell detachment, and Western blotting analysis showed that phosphorylation of FAK and PKC, which were dramatically increased by glucose starvation in HepG2 cells, was markedly suppressed by SNARK.
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PMID:Induction of cell-cell detachment during glucose starvation through F-actin conversion by SNARK, the fourth member of the AMP-activated protein kinase catalytic subunit family. 1457 7

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.
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PMID:Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules. 1603 31

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.
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PMID:Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. 1620 71

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.
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PMID:Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose. 1677 75

Brain selective kinase 2 (BRSK2) has been identified as a member of AMPK related kinases. LKB1 can phosphorylate the Thr174 of BRSK2, increasing its activity >50-fold. In this study, we identified cAMP-dependent protein kinase A (PKA) as another upstream kinase of BRSK2, which can phosphorylate BRSK2 at Thr260. The association between these two proteins was confirmed by GST pull-down. Furthermore, our study indicated that the kinase activity of BRSK2 can be increased through phosphorylation by PKA.
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PMID:BRSK2 is activated by cyclic AMP-dependent protein kinase A through phosphorylation at Thr260. 1687 Jan 37

In an effort to extend the peptide aptamer approach, we have developed a scaffold protein that allows small molecule ligand control over the presentation of a peptide aptamer. This scaffold, a fusion of three protein domains, FKBP12, FRB, and GST, presents a peptide linker region for target protein binding only in the absence of the small molecule Rapamycin or other non-immunosuppressive Rapamycin derivatives. Here we describe the characterization of ligand-regulated peptide aptamers that interact with and inhibit the 5'-AMP-activated protein kinase (AMPK). AMPK, a central regulator of cellular energy homeostasis, responds to high cellular AMP/ATP ratios by promoting energy producing pathways and inhibiting energy consuming biosynthetic pathways. We have characterized 15 LiRPs of similar, poly-basic sequence and have determined that they interact with the substrate peptide binding region of both AMPK alpha1 and alpha2. These proteins, some of which serve as poor substrates of AMPK, inhibit the kinase as pseudosubstrates in a Rapamycin-regulated fashion in vitro, an effect that is largely competitive with substrate peptide and mediated by an increase in the kinase's apparent K(m) for substrate peptide. This pseudosubstrate inhibition of AMPK by LiRP proteins reduced the AMP stimulation of AMPK in vitro and caused the inhibited state of the kinase to kinetically resemble the basal, unstimulated state of AMPK, providing potential insight into the molecular mechanisms of AMP stimulation of AMPK.
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PMID:Ligand-regulated peptide aptamers that inhibit the 5'-AMP-activated protein kinase. 1711 8

Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 mum) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1. Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.
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PMID:Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic beta-cells: evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser517/Ser520 in glucose-stimulated insulin granule movement. 2007 60


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