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*: KSHMKYNRSTKDVK), rat
acetyl-CoA carboxylase (
SAMS: HMRSAMSGLHLVKRR), spinach SPS (SP2: GRRJRRISSVEJJDKK), and spinach
NADH:nitrate reductase (NR6: GPTLKRTASTPFJNTTSK) 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 PKI, PKIV, and PKIII, listed in order of elution. PKIV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca2+ dependent. The novel aspects are that PKIII 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 Ca2+ dependent. Of the three major
kinases that phosphorylated the SP2
peptide only PKI (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 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.