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

Avula Sreenivas,George M. Carman

doi:10.1074/jbc.m300132200

Avula Sreenivas, George M. Carman

Open Access

https://doi.org/10.1074/jbc.m300132200

Copy DOI

Abstract

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-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 UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta 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.

Highlights

Phospholipids are essential molecules that contribute to the structure and function of membranes, and the synthesis of phospholipids is a major activity in which cells engage throughout growth
To determine whether Opi1p was a target for phosphorylation by protein kinase A, we examined whether the kinase catalyzed the incorporation of the ␥ phosphate of 32Plabeled ATP into MBP-Opi1p
The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in membrane phospholipid synthesis in S. cerevisiae (1, 2, 4 – 6)

Summary

EXPERIMENTAL PROCEDURES

For the expression of MBP-Opi1p fusion proteins, the cultures (250 ml) were grown to a cell density of A600 nm ϭ 0.4 – 0.6 at 37 °C, and the cells were harvested by centrifugation at 5,000 ϫ g for 5 min and resuspended in fresh medium containing 0.6 mM isopropyl-␤-D-thiogalactoside. The indicated concentrations of MBP-Opi1p or synthetic peptides were phosphorylated with protein kinase A in a reaction mixture that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.2 mM [␥-32P]ATP (5,000 cpm/pmol), and protein kinase A (0.2 unit/ml). MBP-Opi1p (0.1 mg/ml) was phosphorylated with protein kinase C in a reaction mixture that contained 50 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375 mM EGTA, 1.7 mM CaCl2, 20 ␮M diacylglycerol, 50 ␮M phosphatidylserine, 50 ␮M [␥-32P]ATP (5,000 cpm/pmol), and protein kinase C (1 unit/ml).

Genotype or relevant characteristics

Source or reference

RESULTS

DISCUSSION