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.
Choi
Hyeon-Son
HS
Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
Han
Gil-Soo
GS
Carman
George M
GM
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