Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress
Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress
复制标题
DOI：
10.1074/jbc.m704655200
复制
发表时间：
2007-10-19
影响因子：
4.8
通讯作者：
Shears, Stephen B.
中科院分区：
文献类型：
--
作者：
Choi, Jae H.;Williams, Jason;Shears, Stephen B.
哺乳动物细胞利用多种信号机制来抵御渗透应激，渗透应激伴随着质膜离子运输、溶质摄取以及蛋白质和碳水化合物的转换（Schliess， F., and Haussinger， D., 2002）。化学，383,577-583)。近年来，研究人员发现渗透胁迫可促进高能肌醇焦磷酸二磷酸四磷酸((PP)（2)-InsP(4)）的合成(Pesesse， X., Choi， K., Zhang， T., and Shears， S. B. (2004) J.生物工程学报。化学，279,43378-43381)。在这里，我们描述了从大鼠脑中纯化二磷酸肌醇五磷酸激酶（PPIP5K），该激酶可合成（PP）(2)-InsP(4)。通过质谱分析获得的部分氨基酸序列与含有推测的atp -抓住激酶结构域的160 kda大鼠蛋白序列相匹配。BLAST搜索发现了两个人类亚型PPIP5K1 （160 kDa）和PPIP5K2 （138 kDa）。重组人PPIP5K1在大肠杆菌中表达，可将二磷酸肌醇五磷酸（PP-InsP(5)）磷酸化为（PP）(2)-InsP(4) （V-max = 8.3 nmol/mg protein/min, K-m = 0.34 μ M）。在人胚胎肾细胞中过表达PPIP5K1或PPIP5K2均可显著增加（PP）(2)-InsP(4)的水平，而过表达催化死亡的PPIP5K1（D332A）突变体则没有影响。在体外和体内，PPIP5K1和PPIP5K2对PP-InsP5的活性均高于InsP6。共聚焦免疫荧光分析显示PPIP5K1分布在整个细胞质中，但不在细胞核内。从渗透应激的HEK细胞（0.2 M山梨醇；30分钟）中免疫纯化过表达的PPIP5K1，与对照细胞相比，显示出持续的3.9 +/- 0.4倍的激活。PPIP5Ks可能是重要的信号转导酶。
Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion transport, solute uptake, and turnover of protein and carbohydrates (Schliess, F., and Haussinger, D. (2002) Biol. Chem. 383, 577-583). Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)), a high energy inositol pyrophosphate (Pesesse, X., Choi, K., Zhang, T., and Shears, S. B. (2004) J. Biol. Chem. 279, 43378-43381). Here, we describe the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes (PP)(2)-InsP(4). Partial amino acid sequence, obtained by mass spectrometry, matched the sequence of a 160-kDa rat protein containing a putative ATP-grasp kinase domain. BLAST searches uncovered two human isoforms (PPIP5K1 (160 kDa) and PPIP5K2 (138 kDa)). Recombinant human PPIP5K1, expressed in Escherichia coli, was found to phosphorylate diphosphoinositol pentakisphosphate (PP-InsP(5)) to (PP)(2)-InsP(4) (V-max = 8.3 nmol/mg of protein/min; K-m = 0.34 mu M). Overexpression in human embryonic kidney cells of either PPIP5K1 or PPIP5K2 substantially increased levels of (PP)(2)-InsP(4), whereas overexpression of a catalytically dead PPIP5K1(D332A) mutant had no effect. PPIP5K1 and PPIP5K2 were more active against PP-InsP5 than InsP6, both in vitro and in vivo. Analysis by confocal immunofluorescence showed PPIP5K1 to be distributed throughout the cytoplasm but excluded from the nucleus. Immunopurification of overexpressed PPIP5K1 from osmotically stressed HEK cells (0.2 M sorbitol; 30 min) revealed a persistent, 3.9 +/- 0.4-fold activation when compared with control cells. PPIP5Ks are likely to be important signaling enzymes.