Evidence That Barley 3 - Hydroxy - 3 - Methylglutaryl - Coenzyme A Reductase Kinase 1 s a Member of the Sucrose Nonfermenting - 1 - Related Protein Kinase Family ’

A protein kinase was partially purified from barley (Hordeum vulgare L. cv Sundance) endosperm by ammonium sulfate fractionation, followed by ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatography. It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A (HMC-COA) reductase and a synthetic peptide that was shown previously to act as a substrate for HMC-COA reductase kinase purified from cauliflower, confirming it to be barley HMG-COA reductase kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (M,) of 60,000, which i s the size predicted for the barley sucrose nonfermenting-1 (SNF1)-related protein kinases BKIN2 and BKIN12. Antisera were raised to a rye (Secale cereale 1.) SNF1-related protein kinase (RKIN1) expressed in Fscherichia col i as a fusion with maltosebinding protein and to a synthetic peptide with a sequence that i s conserved in, and specific to, plant members of the SNF1-related protein kinase family. l h e maltose-binding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate M, of 60,000 in crude endosperm extracts and a single polypeptide in root extracts, which co-migrated with the smaller polypeptide in the endosperm doublet. Both antisera recognized a polypeptide with an approximate M, of 60,000 i n the partially purified protein kinase preparation, suggesting strongly that barley HMC-COA reductase kinase is a member of the SNFl-related protein kinase family.