3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE KINASE AND SUCROSE-PHOSPHATE SYNTHASE KINASE-ACTIVITIES IN CAULIFLOWER FLORETS - CA2+ DEPENDENCE AND SUBSTRATE SPECIFICITIES

Plant 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC 1.1.1.34) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) and synthetic peptides designed from the known phosphorylation sites of plant HMGR (SAMS: KS HMKYNRSTKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAMZSGLHLVKRR), sp inach SPS (SP2: GRRJRRISSVEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTASTPFJNTTSK) were used to characterize kinase activities from cauliflower (Brassica oleracea L.) inflorescences, The three majo r peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PKI, PKIV and PKIII listed in order of elution, PKIV was th e most active in terms of phosphorylation and inactivation of recombin ant Nicotiana HMGR and was also strictly Ca2+ dependent. The novel asp ects are that PKIII has not been detected in previous cauliflower stud ies, that SAMS is a more specific peptide substrate to identify poten tial HMGR kinases, and that the major HMGR kinase in cauliflower is Ca 2+ dependent. Of the three major kinases that phosphorylated the SP2 p eptide only PK, (partially Ca2+ sensitive) and PKIII (Ca2+ insensitive ) inactivated native spinach leaf SPS, Cauliflower extracts contained endogenous SPS that was inactivated by endogenous kinase(s) in an ATP- dependent manner and this may be one of the substrate target proteins for PKI and/or PKIII. The substrate specificity of the three kinase pe aks was studied using synthetic peptide variants of the SP2 sequence, All three kinases had a strong preference for peptides with a basic re sidue at P-6 las in SP2 and SAMS; SAMS has a free amino terminus at t his position) or a Pro at P-7 las in NR6), This requirement for certai n residues at P-6 or P-7 was not recognized in earlier studies but app ears to be a general requirement. In plant HMGR, a conserved His resid ue at P-6 is involved directly in catalysis and this may explain why s ubstrates reduced HMGR phosphorylation in vitro. (C) 1998 Academic Pre ss.