The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK coimmunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca 2+ -calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca 2+ -calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaMbinding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.z 1999 Federation of European Biochemical Societies.

The mammalian 5-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (␣ 1 ) is distinct from the previously cloned kinase subunit (␣ 2 ). The ␣ 1 (548 residues) and ␣ 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase ␣ 1 mRNA distribution and the ␣ 1 immunoreactivity rather than ␣ 2 , with substantial amounts in kidney, liver, lung, heart, and brain. Both ␣ 1 and ␣ 2 isoforms are stimulated by AMP and contain noncatalytic ␤ and ␥ subunits. The liver ␣ 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the ␣ 2 immunoreactivity parallels the ␣ 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the ␤ and ␥ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.The 5Ј-AMP-activated protein kinase (AMPK) 1 was initially identified as a protein kinase regulating hydroxymethylglutaryl-CoA reductase (1). Subsequently, the AMPK was shown to phosphorylate hepatic acetyl-CoA carboxylase (2) and adipose hormone-sensitive lipase (3). The AMPK appears to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5Ј-AMP rises in response to fuel limitation and/or hypoxia (4). Partial amino acid sequencing of hepatic AMPK (5, 6) revealed that it consists of 3 subunits, the catalytic subunit ␣ (63 kDa), and two noncatalytic subunits, ␤ (40 kDa) and ␥ (38 kDa).The AMPK is a member of the yeast SNF1 protein kinase subfamily that includes protein kinases present in plants, nematodes, and humans (5-9). The AMPK catalytic subunit, ␣, has strong sequence identity to the catalytic domain of the yeast protein kinase SNF1, which is involved in the induction of invertase (SUC2) under conditions of nutritional stress due to glucose starvation (10). Both Snf1p and the AMPK require phosphorylation by an activating protein kinase for full activity (11). The sequence relationship between Snf1p and AMPK led to the finding that these enzymes share functional similarities, both phosphorylating and inactivating yeast acetyl-CoA carboxylase (5,11,12). Nevertheless, the AMPK does not complement SNF1 in yeast (11), indicating that their full range of functions are not identical. The noncatalytic ␤ and ␥ subunits of AMPK are also related to proteins that interact with Snf1p; the ␤ subunit is related to the SIP1/SIP2/GAL83 family of transcription regulators and the ␥ subunit to SNF4 (also called CAT3) (6, 13). EXPERIMENTAL PROCEDURESPeptide Sequencing-Peptides were derived from rat and porcine ␣ 1 subunit of the AMPK, by in situ proteolysis (5), and sequenced on either an Ap...

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Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

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 ␣ 1 or ␣ 2 catalytic subunits together with ␤ and ␥ noncatalytic subunits in a trimeric complex. The ␣ 1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222- The 5Ј-AMP-activated protein kinase (AMPK) 1 is related to the yeast snf1p subfamily of protein kinases (1) and consists of a catalytic ␣ subunit (63 kDa) and noncatalytic ␤ (40 kDa) and ␥ (38 kDa) subunits. The AMPK phosphorylates a number of key enzymes involved in the control of lipid metabolism, acetylCoA carboxylase, hydroxymethylglutaryl (HMG)-CoA reductase, and hormone-sensitive lipase (2). Because it is activated by elevation of intracellular AMP caused by arsenite and heat shock, it is thought to function primarily in stress responses (3, 4). The activation of the AMPK by AMP results from three contributing mechanisms: direct allosteric activation of the enzyme; AMP activation of an upstream kinase kinase (5, 6); and AMP inhibition of AMPK dephosphorylation (7). Studies on HMG-CoA reductase regulation have reinforced the concept that the AMPK plays a role in stress responses, since mutation of Ser 871 , the AMPK phosphorylation site in HMG-CoA reductase, to Ala blocked phosphorylation by the AMPK and reduction in HMG-CoA reductase activity caused by ATP depletion but did not affect the regulation of HMG-CoA reductase at the transcriptional level (8). In yeast, the snf1p kinase controls invertase transcription in the adaptive response to growth on non-glucose sugars (9) as well as the regulation of acetyl-CoA carboxylase (1, 10), but it is not known whether the AMPK has a similar function.234Recently, we found that multiple isoforms of the AMPK are present in liver (11,12). A distinct gene accounts for ϳ90% of the activity in liver extracts (11). The two isoforms of the AMPK are 90% identical in the catalytic core region and 60% identical in their COOH-terminal tails. Since the AMPK is a multisubstrate protein kinase, its substrate specificity has been of particular interest. Previous studies have shown that an analog of the major AMPK phosphorylation site in rat acetyl-CoA carboxylase, or SAMS peptide , H 73 MRSAMS 79 GLHLVKRR 87 , is a relatively specific substrate for the AMPK. Previous specificity studies performed on the AMPK have used partially purified AMPK likely to have contained a mixture of the ␣ 1 and ␣ 2 isoforms (13-15). Using synthetic peptides based on the SAMS peptide sequence, it was reported that the enzyme has a requirement for two hydrophobic residues at the P ϩ4 and P Ϫ5 positions and a basic residue at either the P Ϫ2,Ϫ3,Ϫ4 site (14). These findings have recently been extended using substitutions in the SAMS peptide variant, AMARAASAAALARR, with partially purified enzyme (15).We have reexamined the specificity of the AMPK in the light of the presence of multiple isoforms of the enzyme. In this paper, we report the p...

The AMP-activated protein kinase (AMPK) consists of catalytic ␣ and noncatalytic ␤ and ␥ subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK ␣ 1 and ␣ 2 catalytic subunits are associated with ␤ 1 and ␥ 1 noncatalytic subunits. We find that the isolated ␥ 1 subunit is N-terminally acetylated with no other posttranslational modification. The 5Ј-AMP-activated protein kinase (AMPK) 1 isolated from liver consists of three subunits, the catalytic ␣ subunit (548 residues, M r 62,497 ϳ63 kDa) and two noncatalytic subunits, ␤ (M r 30,378 ϳ40 kDa) and ␥ (M r 37,429 ϳ38 kDa) (1-3). The AMPK phosphorylates a number of protein substrates including key enzymes involved in the control of lipid metabolism, acetyl-CoA carboxylase, HMG-CoA reductase and hormonesensitive lipase (reviewed in Ref. 4). It has been proposed that the AMPK functions in stress responses since it is activated by increasing intracellular AMP resulting from a variety of treatments including arsenite, heat shock (5-7), ischaemia (8), exercise (9), and electrical stimulation of skeletal muscle (10).There are multiple isoforms of the AMPK subunits present in rat liver (2, 11). The AMPK ␣ 1 isoform (2) is encoded by a gene localized to chromosome 5p11 (12), whereas the AMPK ␣ 2 isoform gene is localized to chromosome 1p31 (13). The ␤ 1 and ␥ 1 subunits genes are located on chromosome 12 (12) The AMPK ␣ 1 and ␣ 2 isoforms are 90% identical in their catalytic cores but only 60% identical in their COOH-terminal tails. Previously, we found that the ␤ 1 subunit of the AMPK was multiply phosphorylated in an intramolecular autophosphorylation reaction (14). The ␤ 1 subunit of the AMPK contains an N-terminal consensus sequence for myristoylation with glycine at position 1 and serine (a small uncharged residue) at position 5 (15).In view of the potential importance of posttranslational modifications of the AMPK subunits in its physiological function, we have characterized the native state of the ␤ subunit. We find that the ␤ subunit purified from rat liver is fully myristoylated and present as a mixture of di-and triphosphorylated species with small amounts of monophospho-␤ subunit. Autophosphorylation of the AMPK in vitro results in up to 6 or more moles of phosphate incorporated per mole of ␤ subunit. The mass of the isolated N-terminally acetylated ␥ subunit is identical to the mass inferred from the cDNA sequence and is not phosphorylated. EXPERIMENTAL PROCEDURESAMPK Purification and Assay-The AMPK was purified from rat liver (14) and assayed (16) as described previously using the SAMS peptide substrate and 200 M 5Ј-AMP. The enzyme was diluted in 50 mM Tris⅐HCl, pH 7.5, 0.05% (v/v) Triton X-100, and the reactions were initiated by adding enzyme. The reactions were stopped by withdrawing 30-l aliquots for liquid scintillation counting as described (17). Protein concentration was assayed by the method of Lowry (18).AMPK Autophosphorylation-Purified AMPK was desalted into assay bu...

A heterotrimeric member of the AMP-activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an K K2 catalytic and two non-catalytic subunits, L L2 and Q Q1. The AMPK L L2 cDNA (271 amino acids (aa), molecular weight (MW) = 30 307, pI 6.3) was cloned from skeletal muscle and found to share an overall identity of 70% with L L1 (270 aa, MW = 30 475, pI 6.0). In the liver AMPK L L1 subunit, Ser-182 is constitutively phosphorylated whereas in skeletal muscle L L2 isoform, we find that Ser-182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser-24, Ser-25 found in the L L1 are replaced by Ala-Glu in the L L2 isoform. L L2 contains seven more Ser and one less Thr residues than L L1, raising the possibility of differential post-translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively L L1 associated with K K2, whereas extensor digitorum longus muscle K K2 (EDL, fast twitch) associates with L L2 as well as L L1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co-immunoprecipitated with the AMPK K K2L L2Q Q1 complex. z 1999 Federation of European Biochemical Societies.

The AMP-activated protein kinase (AMPK) consists of catalytic a and non-catalytic, ß and y (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-OL2 isoform catalytic subunit is associated with ßl and yl and not with ß2 or y2 subunit isoforms. The ßl and yl isoforms are also subunits of the al isoform. The sequence of cloned human AMPK-ßl is 95% identical in amino acid sequence with rat ßl. Human chromosomal localizations were determined for AMPK-al (5pll-pl4), AMPK-ßl (12q24.1-24.3) and AMPK-yl (12ql2-ql4), respectively.