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*: 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.
Toroser
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Department of Crop Science, Agricultural Research Service, Raleigh, North Carolina, 27695-7631, USA.
Huber
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SC
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D001665:Binding Sites; D001937:Brassica; D002118:Calcium / Q000502:physiology*; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D050901:Nitrate Reductase; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D018514:Plant Structures / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D018724:Spinacia oleracea; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D010455:Peptides; D006903:Hydroxymethylglutaryl CoA Reductases; D009565:Nitrate Reductases; D050895:Nitrate Reductase (NADH); D050901:Nitrate Reductase; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D002118:Calcium