Phosphorylation of yeast phosphatidylserine synthase by protein kinase A: identification of Ser46 and Ser47 as major sites of phosphorylation.
The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser(46) --> Ala and/or Ser(47) --> Ala mutations were constructed and expressed in a cho1Delta mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with (32)P(i) and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis, cells expressing the S46A/S47A mutant enzyme exhibited a 57% decrease in phosphatidylserine and a 40% increase in phosphatidylinositol. These results indicate that phosphatidylserine synthase is phosphorylated on Ser(46) and Ser(47) by protein kinase A, which results in a higher amount of enzyme for the net effect of stimulating the synthesis of phosphatidylserine.
Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
Hyeon-Son
HS
Choi
Gil-Soo
GS
Han
George M
GM
Carman
D000483:Alleles; D001665:Binding Sites; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000737:chemistry*; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine / Q000737:chemistry*
D010455:Peptides; D010716:Phosphatidylinositols; D010743:Phospholipids; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase