Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins.
Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.
Moorhead
G
G
Department of Biochemistry, University of Dundee, UK.
Douglas
P
P
Cotelle
V
V
Harthill
J
J
Morrice
N
N
Meek
S
S
Deiting
U
U
Stitt
M
M
Scarabel
M
M
Aitken
A
A
MacKintosh
C
C
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase