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J. Biol. Chem., Vol. 278, Issue 23, 20673-20680, June 6, 2003

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Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A^*

Avula Sreenivas and George M. Carman 

From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901

The Opi1p transcription factor plays a negative regulatory role in the expression of UAS_INO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser³¹ and Ser²⁵¹, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser³¹ and Ser²⁵¹ was examined by measuring the effects of the S31A and S251A mutations on the expression of the UAS_INO-containing gene INO1. The -galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1 mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser³¹ and Ser²⁵¹ mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

Received for publication, January 7, 2003 , and in revised form, March 18, 2003.

^* This work was supported in part by United States Public Health Service Grant GM-50679 from the National Institutes of Health. 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.

To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.

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