Abstract
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.
Highlights
In plants, SAT and OAS-TL are found in multiple subcellular compartments, and the activity of SAT in these compartments is 4 –300-fold less than that of OAS-TL [1,2,3]
In this paper we show that a serine acetyltransferase from soybean (GmSerat2;1) is a substrate of CDPK
Screening of 150,000 plaque-forming units for binding to three 32P-labeled soybean CDPKs resulted in identification of eleven proteins that interacted with CDPK␥
Summary
Plant Material—Soybean cell suspension cultures (Glycine max L.) were maintained as described previously [28]. Expression and Purification of Fusion Proteins—Protocols for expression of recombinant proteins were described previously [35]. Protein Kinase Assay—CDPK activity assays were performed as described previously [31]. Phosphorylation of Serine Acetyltransferase—Phosphorylation of SAT was performed on ice in 50 mM HEPES, pH 7.2, 10 mM MgCl2, 1 mM EGTA, 1.2 mM CaCl2 (omitted for calciumfree reactions), 1 mM DTT, 0.05 mg/ml bovine serum albumin, 1 mM [32P]ATP (ϳ500 cpm/pmol), and 4 ng of His6-CDPK␣ for every 1 g of MBP fusion protein. The reaction was initiated by the addition of [3H]L-serine, incubated for various times at 30 °C, stopped by addition of 0.2 volume of concentrated ammonium hydroxide, and incubated an additional 60 min to ensure complete conversion of OAS to N-acetylserine (NAS) [36]. TLC was performed on Cellulose 300, 100-m TLC plates (Selecto Scientific, Inc., Suwanee, GA) and developed with butanol:water: acetic acid, 65:25:15 by volume
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