14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases.
Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: omega> chi> upsilon>>> phi, psi.
Bachmann
M
M
US Department of Agriculture, Agricultural Research Service, Department of Horticulture, North Carolina State University, Raleigh 27695-7631, USA.
Huber
J L
JL
Athwal
G S
GS
Wu
K
K
Ferl
R J
RJ
Huber
S C
SC
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D004789:Enzyme Activation; D007527:Isoenzymes; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D018724:Spinacia oleracea / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D010940:Plant Proteins; D011506:Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D010749:Phosphoprotein Phosphatases