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PMID: 10318703 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

Plant physiology ·Vol. 120 ·No. 1 ·1999-05-00 ·Pages 257-74

Sugden C, Donaghy PG, Halford NG, Hardie DG

Abstract

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.

MeSH Terms
Amino Acid Sequence Animals Chromatography, Gel Chromatography, Ion Exchange Glucosyltransferases/genetics,metabolism Hydroxymethylglutaryl CoA Reductases/genetics,metabolism Molecular Sequence Data Molecular Weight Nitrate Reductase Nitrate Reductases/genetics,metabolism Peptides/chemistry,metabolism Phosphorylation Plant Leaves/enzymology Protein Serine-Threonine Kinases/genetics,isolation & purification,metabolism Spinacia oleracea/enzymology,genetics Substrate Specificity
Chemicals
Peptides Hydroxymethylglutaryl CoA Reductases Nitrate Reductases Nitrate Reductase Glucosyltransferases sucrose-phosphate synthase SNF1-related protein kinases Protein Serine-Threonine Kinases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Sugden C
Biochemistry Department, Dundee University, Medical Sciences Institute/Wellcome Trust Building Complex, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
Donaghy P G
Halford N G
Hardie D G
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Article Info
Journal
Plant physiology
Abbr.
Plant Physiol
ISSN
0032-0889
DOI
10.1104/pp.120.1.257
Published
1999-05-00
Pages
257-74
Language
English
Region
United States
NLM ID
0401224
PMCID
PMC59258
Subset
IM
Grants
Biotechnology and Biological Sciences Research Council · BBS/E/C/00004150 · United Kingdom
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1104/pp.120.1.257 PMC Full Text View journal details
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