Calcium-regulated phosphorylation of soybean serine acetyltransferase in response to oxidative stress.
Glycine max serine acetyltransferase 2;1 (GmSerat2;1) is a member of a family of enzymes that catalyze the first reaction in the biosynthesis of cysteine from serine. It was identified by interaction cloning as a protein that binds to calcium-dependent protein kinase. In vitro phosphorylation assays showed that GmSerat2;1, but not GmSerat2;1 mutants (S378A or S378D), were phosphorylated by soybean calcium-dependent protein kinase isoforms. Recombinant GmSerat2;1 was also phosphorylated by soybean cell extract in a Ca2+-dependent manner. Phosphorylation of recombinant GmSerat2;1 had no effect on its catalytic activity but rendered the enzyme insensitive to the feedback inhibition by cysteine. In transient expression analyses, fluorescently tagged GmSerat2;1 localized in the cytoplasm and with plastids. Phosphorylation state-specific antibodies showed that an increase in GmSerat2;1 phosphorylation occurred in vivo within 5 min of treatment of soybean cells with 0.5 mM hydrogen peroxide, whereas GmSerat2;1 protein synthesis was not significantly induced until 1 h after oxidant challenge. Internal Ca2+ was required in the induction of both GmSerat2;1 phosphorylation and synthesis. Treatment of cells with calcium antagonists showed that externally derived Ca2+ was important for retaining GmSerat2;1 at a basal level of phosphorylation but was not necessary for its hydrogen peroxide-induced synthesis. Protein phosphatase type 1, but not type 2A or alkaline phosphatase, dephosphorylated native GmSerat2;1 in vitro. These results support the hypothesis that GmSerat2;1 is regulated by calcium-dependent protein kinase phosphorylation in vivo and suggest that increased GmSerat2;1 synthesis and phosphorylation in response to active oxygen species could play a role in anti-oxidative stress response.
Liu
Fenglong
F
Program in Plant Molecular and Cellular Biology and the Department of Botany, University of Florida, Gainesville, Florida 32611-8526, USA.
Yoo
Byung-Chun
BC
Lee
Jung-Youn
JY
Pan
Wei
W
Harmon
Alice C
AC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D005816:Genetic Complementation Test; D006861:Hydrogen Peroxide / Q000737:chemistry; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010766:Phosphorylation; D020033:Protein Isoforms; D017422:Sequence Analysis, DNA; D051050:Serine O-Acetyltransferase / Q000737:chemistry*; D013025:Glycine max / Q000201:enzymology*
D020033:Protein Isoforms; D006861:Hydrogen Peroxide; D051050:Serine O-Acetyltransferase; D002118:Calcium