Gu Chunfang, Nguyen Hoai-Nghia, Hofer Alexandre, Jessen Henning J, Dai Xuming, Wang Huanchen, Shears Stephen B
From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, 27709.
Department of Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
J Biol Chem. 2017 Mar 17;292(11):4544-4555. doi: 10.1074/jbc.M116.765743. Epub 2017 Jan 26.
Proteins responsible for P homeostasis are critical for all life. In , extracellular [P] is "sensed" by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP) as follows: during a period of P starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) 42, 219-231. Hitherto, such P sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP and ATP decrease upon [P] starvation and subsequently recover during P replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP), reacts more dramatically ( with a wider dynamic range and greater sensitivity). To understand this novel InsP response, we characterized kinetic properties of the bifunctional 5-InsP kinase/InsP phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP in this case) to fluctuate independently of varying precursor ( 5-InsP) pool size. Moreover, we report that InsP phosphatase activities of PPIP5Ks are strongly inhibited by P (40-90% within the 0-1 mm range). For PPIP5K2, P sensing by InsP is amplified by a 2-fold activation of 5-InsP kinase activity by P within the 0-5 mm range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling, regulating InsP turnover independently of 5-InsP, in response to fluctuations in extracellular supply of a key nutrient.
负责磷（P）体内平衡的蛋白质对所有生命都至关重要。在（某种情况中），细胞外的[P]由肌醇六磷酸激酶（IP6K）“感知”，该激酶合成细胞内肌醇焦磷酸5-二磷酸肌醇1,2,3,4,6-五磷酸（5-InsP），具体过程如下：在磷饥饿期间，细胞内[ATP]下降；IP6K对ATP异常低的亲和力迫使5-InsP水平随之下降（阿泽维多，C.，和赛亚尔迪，A.（2017年）《（文献名未给出）》42卷，219 - 231页）。迄今为止，后生动物中尚未有此类磷感知的记录。在此，我们使用人肠上皮细胞系（HCT116）表明，在[P]饥饿时5-InsP和ATP水平均下降，随后在补充磷时恢复。然而，另一种肌醇焦磷酸，1,5-双二磷酸肌醇2,3,4,6-四磷酸（InsP），反应更为显著（具有更宽的动态范围和更高的灵敏度）。为了解这种新的InsP反应，我们表征了全长二磷酸肌醇五磷酸激酶（PPIP5K）的双功能5-InsP激酶/InsP磷酸酶活性的动力学特性。这些数据满足了先前发表的任何双功能激酶/磷酸酶表现出浓度稳健性的标准，使激酶产物（在这种情况下为InsP）的水平能够独立于不同的前体（5-InsP）库大小而波动。此外，我们报告PPIP5K的InsP磷酸酶活性受到磷的强烈抑制（在0 - 1毫米范围内抑制40 - 90%）。对于PPIP5K2，在0 - 5毫米范围内，磷对5-InsP激酶活性的2倍激活放大了InsP对磷的感知。总体而言，我们的数据揭示了有助于肌醇焦磷酸信号传导特异性的机制，可独立于5-InsP调节InsP周转，以响应关键营养素细胞外供应的波动。
