Ken I. Mitchelhill

AMP-activated protein kinase phosphorylation of endothelial NO synthase

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 co‐immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser‐1177 in the presence of Ca2+‐calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+‐calmodulin, AMPK also phosphorylates eNOS at Thr‐495 in the CaM‐binding 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.

Dealing with energy demand: the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes. AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia/hypoxia, and modulates many aspects of mammalian cell metabolism. The AMPK yeast homolog, Snf1p, plays a major role in adaption to glucose deprivation. In mammals, AMPK also has diverse roles that extend from energy metabolism through to transcriptional control.

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.

Posttranslational Modifications of the 5′-AMP-activated Protein Kinase β1 Subunit

The AMP-activated protein kinase (AMPK) consists of catalytic α and noncatalytic β and γ 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 α1 and α2 catalytic subunits are associated with β1 and γ1 noncatalytic subunits. We find that the isolated γ1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated β1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable β1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated β1subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylatedin vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that α1 Thr172 is a major, although not exclusive, site of both basal and stimulated α1phosphorylation by an upstream AMPK kinase.

Expression of the AMP-activated protein kinase β1 and β2 subunits in skeletal muscle

A heterotrimeric member of the AMP‐activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an α2 catalytic and two non‐catalytic subunits, β2 and γ1. The AMPK β2 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 β1 (270 aa, MW=30 475, pI 6.0). In the liver AMPK β1 subunit, Ser‐182 is constitutively phosphorylated whereas in skeletal muscle β2 isoform, we find that Ser‐182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser‐24, Ser‐25 found in the β1 are replaced by Ala‐Glu in the β2 isoform. β2 contains seven more Ser and one less Thr residues than β1, raising the possibility of differential post‐translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively β1 associated with α2, whereas extensor digitorum longus muscle α2 (EDL, fast twitch) associates with β2 as well as β1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co‐immunoprecipitated with the AMPK α2β2γ1 complex.

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP‐activated protein kinase (AMPK) consists of catalytic α and non‐catalytic, β and γ (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‐α2 isoform catalytic subunit is associated with β1 and γ1 and not with β2 or γ2 subunit isoforms. The β1 and γ1 isoforms are also subunits of the α1 isoform. The sequence of cloned human AMPK‐β1 is 95% identical in amino acid sequence with rat β1. Human chromosomal localizations were determined for AMPK‐α1 (5p11‐p14), AMPK‐β1 (12q24.1‐24.3) and AMPK‐γ1 (12q12‐q14), respectively.

Phosphorylation at the Cyclin-dependent Kinases Site (Thr85) of Parathyroid Hormone-related Protein Negatively Regulates Its Nuclear Localization

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61–94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10, 34, 35) for PTHrP.