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

Dikran Toroser, Steven C. Huber

Research output: Contribution to journalArticlepeer-review

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*: 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 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 Ca2+ 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 Ca2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PK(I) (partially Ca2+ sensitive) and PK(III) (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 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.

Original languageEnglish (US)
Pages (from-to)291-300
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume355
Issue number2
DOIs
StatePublished - Jul 15 1998

Keywords

  • 3-hydroxy-3-methylglutaryl-CoA reductase
  • Brassica oleracea
  • Phosphorylation motif
  • Protein kinase
  • Sucrose-phosphate synthase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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