Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
J Biol Chem. 2010 Apr 9;285(15):11526-36. doi: 10.1074/jbc.M110.100727. Epub 2010 Feb 9.
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.
酵母细胞 CHO1 编码的磷酸丝氨酸合成酶可在体外被蛋白激酶 A 磷酸化并抑制。构建了 CHO1 等位基因 Ser(46) --> Ala 和/或 Ser(47) --> Ala 突变，并在缺乏磷酸丝氨酸合成酶的 cho1Delta 突变体中表达。体外，S46A/S47A 突变使磷酸化总量减少 90%，并消除了蛋白激酶 A 对磷酸丝氨酸合成酶活性的抑制作用。蛋白激酶 A 对磷酸丝氨酸合成酶的磷酸化作用是时间和剂量依赖性的，且依赖于 ATP 的浓度，导致其电泳迁移率从 27 kDa 形式变为 30 kDa 形式。磷酸丝氨酸合成酶的两种电泳形式存在于指数期细胞中，而只有 27 kDa 形式存在于静止期细胞中。用 (32)P(i) 进行体内标记和免疫复合物分析表明，与 27 kDa 形式相比，30 kDa 形式主要被磷酸化。然而，S46A/S47A 突变消除了蛋白激酶 A 介导的电泳迁移率变化。S46A/S47A 突变还导致指数期细胞中磷酸丝氨酸合成酶的总含量减少 55%，静止期细胞中酶的含量减少 66%。在磷脂组成分析中，表达 S46A/S47A 突变酶的细胞中，磷酸丝氨酸减少 57%，磷脂酰肌醇增加 40%。这些结果表明，磷酸丝氨酸合成酶在 Ser(46)和 Ser(47)上被蛋白激酶 A 磷酸化，这导致更多的酶用于刺激磷酸丝氨酸合成的净效应。
