Characterization of the substrate specificity of sucrose-phosphate synthase protein kinase.
Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated by reversible protein phosphorylation. When the enzyme is phosphorylated it is inactivated and can be reactivated by removal of phosphate. The major regulatory phosphorylation site is known to be Ser158 in the spinach-leaf enzyme, and two protein kinase activities have been resolved chromatographically which phosphorylate SPS at this site in vitro. In this report, we use a set of synthetic peptide substrate analogs based on the phosphorylation site sequence, and a set of Escherichia coli-expressed 26-kDa fragments of spinach SPS which contain the site, to identify the recognition elements that target the two protein kinases to Ser158. The major recognition element consists of basic residues at P-3 and P-6 relative to the phosphorylated serine. Comparison of the spinach enzyme amino-acid sequence with two other plant species show conservation of these amino acids and implies that these signals are also conserved. We also present evidence that glucose-6-phosphate is not only an allosteric activator of SPS but also an inhibitor of SPS-protein kinase per se, thereby allowing it to act at both levels of SPS regulation.
McMichael
R W
RW
U.S. Department of Agriculture, North Carolina State University, Raleigh 27695-7631, USA.
Kochansky
J
J
Klein
R R
RR
Huber
S C
SC
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity
D010455:Peptides; D011994:Recombinant Proteins; D010768:Phosphoserine; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D011494:Protein Kinases