3-Hydroxy-3-methylglutaryl-coenzyme A reductase kinase and sucrose-phosphate synthase kinase activities in cauliflower florets: Ca2+ dependence and substrate specificities.

Abstract 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*: KSHMKYNR S TKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAM S GLHLVKRR), spinach SPS (SP2: GRRJRRIS S VEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTA S TPFJNTTSK) were used to characterize kinase activities from cauliflower ( Brassica oleracea L.) inflorescences. The three major peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PK I , PK IV , and PK III , listed in order of elution. PK IV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca 2+ dependent. The novel aspects are that PK III has not been detected in previous cauliflower studies, that SAMS* is a more specific peptide substrate to identify potential HMGR kinases, and that the major HMGR kinase in cauliflower is Ca 2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PK I (partially Ca 2+ sensitive) and PK III (Ca 2+ 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 PK I and/or PK III . The substrate specificity of the three kinase peaks was studied using synthetic peptide variants of the SP2 sequence. All three kinases had a strong preference for peptides with a basic residue at P-6 (as in SP2 and SAMS*; SAMS has a free amino terminus at this position) or a Pro at P-7 (as in NR6). This requirement for certain residues at P-6 or P-7 was not recognized in earlier studies but appears to be a general requirement. In plant HMGR, a conserved His residue at P-6 is involved directly in catalysis and this may explain why substrates reduced HMGR phosphorylation in vitro.