Pah1磷脂酸(PA)磷酸酶通过产生其前体二酰基甘油在酿酒酵母的三酰基甘油合成中起主要作用,并通过消耗其前体PA同时调节从头磷脂的合成。Pah1的功能需要它的膜定位,由其磷酸化状态控制。Pah1被Nem1-Spo7蛋白磷酸酶去磷酸化,而其磷酸化发生于多种已知和未知的蛋白激酶。在这项工作中,我们表明Rim11是哺乳动物糖原合成酶激酶-3β的酵母同源物,是在丝氨酸(Ser12、Ser602和Ser818)和苏氨酸(Thr163、Thr164、Thr522)残基上磷酸化Pah1的蛋白激酶。Rim11的酶学特征表明,其对Pah1的Km(0.4μM)与其他Pah1磷酸化蛋白激酶的Km相似,但是ATP的Km(30μM)明显高于这些相同激酶的Km。此外,我们证明了Pah1的Rim11磷酸化不需要底物预磷酸化,但在Pho85-Pho80蛋白激酶预磷酸化后增加了~2倍。此外,我们显示Rim11磷酸化的Pah1是Nem1-Po7去磷酸化的底物。最后,我们证明了Pah1的Rim11磷酸化通过降低其催化效率对其PA磷酸酶活性产生抑制作用。主要磷酸化位点(Thr163,Thr164和Ser602)的突变分析表明,需要Rim11介导的这些位点的磷酸化来确保Nem1-Po7依赖性酶在膜上的定位。总的来说,这些发现促进了我们对磷酸化介导的脂质合成中Pah1功能调节的理解.
Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that
Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of
Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate
Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show
Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that
Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.