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J. Biol. Chem., Vol. 281, Issue 37, 27405-27415, September 15, 2006

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Calcium-regulated Phosphorylation of Soybean Serine Acetyltransferase in Response to Oxidative Stress^*

Fenglong Liu¹, Byung-Chun Yoo², Jung-Youn Lee²³, Wei Pan, and Alice C. Harmon⁴

From the Program in Plant Molecular and Cellular Biology and the Department of Botany, University of Florida, Gainesville, Florida 32611-8526 and the Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711

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.

Received for publication, May 11, 2006 , and in revised form, July 17, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) AY422685 [GenBank] .

^* This work was supported in part by National Science Foundation Grant MCB-9604647 (to A. C. H.) and by funds from the University of Florida (to A. C. H.) and from the University of Delaware and the College of Agriculture and Natural Resources Research Partnership (to B.-C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

This article was selected as a Paper of the Week.

¹ Current address: Dept. of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115.

² Current address: Crop Genetics Research, E.I. du Pont de Nemours and Company, Wilmington, DE 19880-0353.

³ Supported by National Institutes of Health Grant P20 RR-15588 from the National Center for Research Resources.

⁴ To whom correspondence should be addressed: Cancer/Genetics Complex, University of Florida, P. O. Box 103610, Gainesville, FL 32610-3610. Tel.: 352-273-8096; Fax: 352-392-3993; E-mail: harmon{at}ufl.edu.

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