Two SNF1-Related Protein Kinases from Spinach Leaf Phosphorylate and Inactivate 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, Nitrate Reductase, and Sucrose Phosphate Synthase in Vitro

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme Areductase kinase A andC) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related proteinkinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides. AMPKAMP-activated protein kinaseAMPKKAMPK kinaseHMG3-hydroxy-3-methylglutarylHRKHMG-CoA reductase kinaseNRnitrate reductasePP2Aprotein phosphatase-2APP2Cprotein phosphatase-2CSPSSuc phosphate synthaseTFAtrifluoroacetic acid