Frosa Katsis

Coordinated Control of Endothelial Nitric-oxide Synthase Phosphorylation by Protein Kinase C and the cAMP-dependent Protein Kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.

Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase.

The 5′-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either α1 or α2 catalytic subunits together with β and γ noncatalytic subunits in a trimeric complex. The α1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK α1 with a Km of 3.8 μM and a Vmax of 4.8 μmol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 μM and a Vmax of 8.1 μmol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK α1 isoform has a Kcat ∼250-fold higher than the AMPK α2 isoform isolated from rat liver. The AMPK α1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Φ(X, β)XXS/TXXXΦ (Φ, hydrophobic; β, basic). In good AMPK α1 peptide substrates, a hydrophobic residue at the P−5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.

AMP-activated Protein Kinase β Subunit Tethers α and γ Subunits via Its C-terminal Sequence (186–270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable αβγ heterotrimer comprising a catalytic α and two non-catalytic subunits, β and γ. The β subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the β subunit, β1-(186–270), is sufficient to form an active AMP-dependent heterotrimer α1β1-(186–270)-γ1, whereas the 25-residue β1 C-terminal (246–270) sequence is sufficient to bind γ1, γ2, or γ3 but not the α subunit. Deletion of the β C-terminal Ile-270 precludes βγ association in the absence of the α subunit, but the presence of the α subunit or substitution of Ile-270 with Ala or Glu restores βγ binding. Truncation of the α subunit reveals that β1 binding requires the α1-(313–473) sequence. The conserved C-terminal 85-residue sequence of the β subunit (90% between β1 and β2) is the primary αγ binding sequence responsible for the formation of the AMPK αβγ heterotrimer.

AMPK beta subunit tethers alpha and gamma subunits via its C-terminal sequence(186-270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable αβγ heterotrimer comprising a catalytic α and two non-catalytic subunits, β and γ. The β subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the β subunit, β1-(186–270), is sufficient to form an active AMP-dependent heterotrimer α1β1-(186–270)-γ1, whereas the 25-residue β1 C-terminal (246–270) sequence is sufficient to bind γ1, γ2, or γ3 but not the α subunit. Deletion of the β C-terminal Ile-270 precludes βγ association in the absence of the α subunit, but the presence of the α subunit or substitution of Ile-270 with Ala or Glu restores βγ binding. Truncation of the α subunit reveals that β1 binding requires the α1-(313–473) sequence. The conserved C-terminal 85-residue sequence of the β subunit (90% between β1 and β2) is the primary αγ binding sequence responsible for the formation of the AMPK αβγ heterotrimer.

Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.

Identification of a parathyroid hormone in the fish Fugu rubripes

A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1–34) had 5‐fold lower potency than human PTH(1–34) in a mammalian cell system.

Evidence That the Pertussis Toxin-sensitive Trimeric GTP-binding Protein Gi2 Is Required for Agonist- and Store-activated Ca2+ Inflow in Hepatocytes

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein α subunits, and carboxyl-terminal α-subunit synthetic peptides. An anti-Gi1−2α antibody and a Gi2α peptide (Gi2α Ile345-Phe355), but not a Gi3α peptide (Gi3α Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gqα antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2α is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11α was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.

Phosphorylation of Neuronal and Endothelial Nitric Oxide Synthase in the Kidney with High and Low Salt Diets

Background: Renal nitric oxide (NO) synthesis increases with increasing salt intake, however, the mechanisms underlying this are poorly understood. We hypothesized that activating or inhibitory phosphorylation of neuronal and endothelial nitric oxide synthase (nNOS, eNOS) regulates renal NO production in response to altered dietary salt. Methods:Sprague-Dawley rats were fed low, normal or high salt diets for 12 h or 2 weeks, and kidney NOS phosphorylation was analyzed by Western blot using phosphopeptide antibodies against the sites nNOS-Ser1412, nNOS-Ser847, eNOS-Ser1176 and eNOS-Thr494. Results:At 12 h, total nNOS increased 1.4-fold (p < 0.01) in the high salt group and decreased by 26% (p < 0.05) in the low salt group. Changes in expression of phospho-nNOS at 12 h were accounted for by the changes in total nNOS. No change in total or phospho-eNOS was seen at 12 h. At 2 weeks, in the low salt group expression of total nNOS increased 1.8-fold (p < 0.05) whereas expression of nNOS phosphorylated at the inhibitory site Ser847 increased 4.3-fold (p < 0.01). Total eNOS was increased 3-fold in the low salt group (p < 0.01), with parallel increases in eNOS phosphorylated at both activating and inhibitory sites (p < 0.05). In the 2-week high salt group no changes in NOS expression or phosphorylation were seen, despite the observed increased excretion of urinary NO metabolites. Conclusion:In summary, changes in phospho-nNOS and phospho-eNOS expression occurred in parallel with changes in total expression, thus, the overall activating and inhibitory effects of nNOS and eNOS phosphorylation at the sites studied were not changed by altered dietary salt.

AMP-activated Protein Kinase Subunit Interactions β1:γ1 ASSOCIATION REQUIRES β1 Thr-263 AND Tyr-267

AMP-activated protein kinase (AMPK) plays multiple roles in the bodys overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic α subunit and two non-catalytic subunits, β and γ, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK α1β1γ1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian αβγ complex shows that β binds α and γ. Here we show that a β1(186–270)γ1 complex can form in the absence of detectable α subunit. Moreover, using alanine mutagenesis we show that β1 Thr-263 and Tyr-267 are required for βγ association but not αβ association.

AMPK subunit interactions; β1:γ1 association requires β1 THR-263 and TYR-267

AMP-activated protein kinase (AMPK) plays multiple roles in the bodys overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic α subunit and two non-catalytic subunits, β and γ, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK α1β1γ1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian αβγ complex shows that β binds α and γ. Here we show that a β1(186–270)γ1 complex can form in the absence of detectable α subunit. Moreover, using alanine mutagenesis we show that β1 Thr-263 and Tyr-267 are required for βγ association but not αβ association.