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Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A
Author(s) -
Choi HyeonSon,
Carman George M.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.689.3
Subject(s) - biochemistry , phosphorylation , phosphatase , protein kinase a , protein phosphorylation , autophagy related protein 13 , microbiology and biotechnology , enzyme , biology , proteolysis , atp synthase , c raf , chemistry , mitogen activated protein kinase kinase
Phosphatidylserine (PS) synthase catalyzes the committed step of phosphatidylcholine synthesis via the CDP‐diacylglycerol pathway in the yeast Saccharomyces cerevisiae . It is one of the most highly regulated phospholipid synthesis enzymes in yeast. Previous work showed that PS synthase is phosphorylated by protein kinase A, and that phosphorylation inhibited enzyme activity. In this work, the sites of protein kinase A phosphorylation were examined. Upon SDS‐PAGE, the PS synthase migrates as a doublet of two protein bands. Protein kinase A phosphorylation of the native enzyme resulted in the disappearance of the lower protein band, whereas treatment with alkaline phosphatase or protein phosphatase resulted in the disappearance of the upper protein band. A site‐directed mutagenesis analysis of putative protein kinase A sites (Ser 46 , Ser 47 , Ser 140 , Ser 141 ) revealed that Ser46 and Ser47 were the major phosphorylation sites in the enzyme. A double S46A S47A mutation resulted in an enzyme that migrated on SDS‐gels as a single protein band, whereas single S46A and S47A mutant proteins migrated as doublets with a decrease in the abundance of the upper band. PS synthase was also subject to proteolytic degradation, and the phosphorylation of the enzyme at Ser46 and Ser47 protected the enzyme from proteolysis. Supported by NIH grant GM 50679.