Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.
The intact, 100 kd microsomal enzyme and the 53 kd catalytic fragment of rat HMG-CoA reductase are both phosphorylated and inactivated by the AMP-activated protein kinase. Using the catalytic fragment, we have purified and sequenced peptides containing the single site of phosphorylation. Comparison with the amino acid sequence predicted from the cDNAs encoding other mammalian HMG-CoA reductases identifies this site as a serine residue close to the C-terminus (Ser872 in the human enzyme). Phosphopeptide mapping of native, 100 kd microsomal HMG-CoA reductase confirms that this C-terminal serine is the only major site phosphorylated in the intact enzyme by the AMP-activated protein kinase. The catalytic fragment of HMG-CoA reductase was also isolated from rat liver in the presence of protein phosphatase inhibitors under conditions where the enzyme is largely in the inactive form. HPLC, mass spectrometry and sequencing of the peptide containing Ser872 demonstrated that this site is highly phosphorylated in intact liver under these conditions. We have also identified by amino acid sequencing the N-terminus of the catalytic fragment, which corresponds to residue 423 of the human enzyme.
Clarke
P R
PR
Protein Phosphorylation Group, Department of Biochemistry, The University, Dundee, Scotland, UK.
Hardie
D G
DG
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003488:Cyanogen Bromide; D006706:Homeostasis; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011817:Rabbits; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D009097:Multienzyme Complexes; D010748:Phosphopeptides; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D003488:Cyanogen Bromide