Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.
Experiments were performed to investigated the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca(2+)-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca(2+)-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca(2+)-dependent, 150-kD protein kinase.
Toroser
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United States Department of Agriculture, Agricultural Research Service, Raleigh, North Carolina 27695-7631 USA.
Huber
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SC
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D003624:Darkness; D004789:Enzyme Activation; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine; D018515:Plant Leaves; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology* / Q000502:physiology
D010446:Peptide Fragments; D010748:Phosphopeptides; D010768:Phosphoserine; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D011494:Protein Kinases; D002118:Calcium