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 alpha(1) or alpha(2) catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex, The alpha(1) isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P-229 (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 Ser(230) by AMPK alpha(1) with a K-m of 3.8 mu M and a V-max of 4.8 mu mol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A(77)R(86-87) peptide substrate, HMRSAMSGLHLVKRR, with a K-m of 33.3 mu M and a V-max of 8.1 mu mol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha(1) isoform has a K-cat similar to 250-fold higher than the AMPK alpha(2) isoform isolated from rat liver. The AMPK alpha(1) 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 Phi(X, beta)XXS/TXXX Phi (Phi, hydrophobic; beta, basic), In good AMPK alpha(1) 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)A(77)R(86-87) peptide increased the K-m 6-fold and reduced the V-max to 4% of the reduced peptide.