EVIDENCE THAT BARLEY 3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A REDUCTASE KINASE IS 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 b y ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatogr aphy. It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglut aryl-coenzyme A (HMG-CoA) reductase and a synthetic peptide that was s hown previously to act as a substrate for HMG-CoA reductase kinase pur ified from cauliflower, confirming it to be barley HMG-CoA reductase k inase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of th e partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (M(r)) of 60,000, which is the size predicted for the barley sucrose nonfermenting-l (SNF1)-re lated protein kinases BKIN2. and BKIN12. Antisera were raised to a rye (Secale cereale L.) SNF1-related protein kinase (RKIN1) expressed in Escherichia coli as a fusion with maltose-binding protein and to a syn thetic peptide with a sequence that is conserved in, and specific to, plant members of the SNF1-related protein kinase family. The maltose-b inding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate M(r) of 60,000 in crude endosperm ext racts and a single polypeptide in root extracts, which co-migrated wit h the smaller polypeptide in the endosperm doublet. Both antisera reco gnized a polypeptide with an approximate M(r) of 60,000 in the partial ly purified protein kinase preparation, suggesting strongly that barle y HMG-CoA reductase kinase is a member of the SNF1-related protein kin ase family.