Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation*
Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation*
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DOI：
10.1074/jbc.m409457200
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发表时间：
2005-03
影响因子：
4.8
通讯作者：
T. Yanagawa;T. Funasaka;S. Tsutsumi;Tirza Raz;N. Tanaka;A. Raz
中科院分区：
文献类型：
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作者：
T. Yanagawa;T. Funasaka;S. Tsutsumi;Tirza Raz;N. Tanaka;A. Raz
磷酸葡萄糖异构酶（PGI; EC 5.3.1.9）是糖代谢途径的细胞溶质管家酶，其在糖酵解和糖异生中起关键作用。PGI是一种多功能的二聚体蛋白，其在细胞外充当细胞因子，具有包括自分泌运动因子（AMF）-引发促有丝分裂、促运动和分化功能的特性，并且PGI已经涉及肿瘤进展和转移。尽管已知人PGI/AMF通过蛋白激酶CK 2（CK 2）在Ser 185处磷酸化，但对PGI/AMF活性的生化调节知之甚少;然而，这种磷酸化的生理意义尚不清楚。因此，通过定点诱变，我们用天冬氨酸（S185 D）或谷氨酸（S185 E）取代Ser 185，这引入了模拟磷酸化的负电荷和构象变化。产生Ser-to-Ala突变蛋白（S185 A）以消除磷酸化。生化分析表明，磷酸化突变蛋白的PGI表现出降低酶活性，而S185 A突变PGI蛋白保留完整的酶活性。CK 2引起的PGI磷酸化也导致酶活性下调。此外，通过RNA干扰敲低CK 2与细胞PGI酶活性的上调相关。与野生型蛋白相比，三种重组突变蛋白表现出难以区分的细胞因子活性和受体结合亲和力。在体外和体内测定中，野生型和S185 A突变蛋白经历活性物质二聚化，而S185 D和S185 E突变蛋白也形成四聚体。这些结果表明磷酸化影响酶的变构动力学性质，导致PGI的活性较低形式，而非磷酸化蛋白质物质保留细胞因子活性。因此，磷酸化调节PGI酶活性的过程对于理解这种关键的葡萄糖代谢调节酶的生物调节具有重要意义。
Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser185 by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.