ISOFORM-SPECIFIC PURIFICATION AND SUBSTRATE-SPECIFICITY OF THE 5'-AMP-ACTIVATED PROTEIN-KINASE

The 5'-AMP-activated protein kinase (AMPK) mediates several cellular r esponses to metabolic stress, Rat liver contains at least two isoforms of this enzyme, either alpha(1) or alpha(2) catalytic subunits togeth er with beta and gamma noncatalytic subunits in a trimeric complex, Th e alpha(1) isoform is purified using a peptide substrate affinity chro matography column with ADR1 (222-234)P-229 (LKKLTRRPSFSAQ), correspond ing to the cAMP-dependent protein kinase phosphorylation site in the y east 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 an d a V-max of 4.8 mu mol/min/mg compared to the commonly used rat acety l-CoA carboxylase (73-87)A(77)R(86-87) peptide substrate, HMRSAMSGLHLV KRR, with a K-m of 33.3 mu M and a V-max of 8.1 mu mol/min/mg. Thus, t he AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha(1) isoform has a K-cat simila r to 250-fold higher than the AMPK alpha(2) isoform isolated from rat liver. The AMPK alpha(1) isoform readily phosphorylates peptides corre sponding to the reported AMPK phosphorylation sites in rat, chicken, a nd yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA redu ctase but not phosphorylase kinase, Based on previous peptide substrat e specificity studies (Dale, S., Wilson W. A., Edelman, A. M., and Har die, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzym e and variants of the peptide AMARAASAAALARRR, it was proposed that th e AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXX P hi (Phi, hydrophobic; beta, basic), In good AMPK alpha(1) peptide subs trates, 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.