Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli

Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism.     

Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr residue

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.     

The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.     

Ligand-regulated peptide aptamers that inhibit the 5'-AMP-activated protein kinase

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.     

M-LDH serves as a regulatory subunit of the cytosolic substrate-channelling complex in vivo

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.     

AICAR induces phosphorylation of AMPK in an ATM-dependent, LKB1-independent manner

AMPK is an AMP-activated protein kinase that plays an important role in regulating cellular energy homeostasis. Metabolic stress, such as heat shock and glucose starvation, causes an energy deficiency in the cell and leads to elevated levels of intracellular AMP. This results in the phosphorylation and activation of AMPK. LKB1, a tumor suppressor, has been identified as an upstream kinase of AMPK. We found that in response to treatment with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), the LKB1 deficient cancer cell line, HeLa, exhibited AMPK-α phosphorylation. This indicates the existence of an LKB1-independent AMPK-α phosphorylation pathway. ATM is a protein that is deficient in the disease ataxia telangiectasia (A-T). We measured the activation of AMPK by AICAR in the normal mouse embryo fibroblast cell line, A29, and the mouse cell line lacking the ATM protein, A38. In A38 cells, the level of AICAR-induced AMPK-α phosphorylation was significantly lower than that found in A29 cells. Furthermore, phosphorylation of AMPK in HeLa and A29 cells was inhibited by an ATM specific inhibitor, KU-55933. Our results demonstrate that AICAR treatment could lead to phosphorylation of AMPK in an ATM-dependent and LKB1-independent manner. Thus, ATM may function as a potential AMPK kinase in response to AICAR treatment.     

Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα.