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.