Phosphatidylinositol 5-phosphate biosynthesis is linked to PIKfyve and is involved in osmotic response pathway in mammalian cells
Phosphatidylinositol 5-phosphate biosynthesis is linked to PIKfyve and is involved in osmotic response pathway in mammalian cells
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DOI：
10.1074/jbc.m207576200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Sbrissa, D;Ikonomov, OC;Shisheva, A
磷脂酰肌醇(PtdIns)5-P是磷脂酰肌醇(PI)家族的最新成员，其细胞功能、调节和酶学仍不清楚。虽然已经指定了一种使用PtdIns-5-P作为细胞内底物的激酶，但产生它的激酶仍有待鉴定。在此，我们报道了PIKfyve，一种在体外合成PtdIns-5-P和在体外和体内合成PtdIns-3，5-P-2的酶，在细胞环境中负责PtdIns-5-P的产生。证据基于两种独立的方法对两组细胞类型的检查。首先，[P-32]正磷酸盐标记的细胞(Sf9、3T3-L1成纤维细胞和3T3-L1脂肪细胞)在基础条件下显示PtdIns-5-P组可检测到的高压液相色谱峰显示，当PIK-Fyve(WT)表达时，放射性PtdIns-5-P的量增加了20%-50%。其次，在细胞类型(HEK293)中，当诱导表达PIKfyveWT时，在体外II型PIP激酶的诱导下，内源性PtdIns-5-P池转化为PtdIns-4，5-P-2的能力较高，其中放射性PtdIns-5-P的基础水平经HPLC头基团分析无法检测到。相反，PtdIns-5-P到PtdIns-4，5-P-2的转化率下降60%与诱导HEK293细胞中显性负性激酶缺陷的PIKfyve(K1831E)突变体的表达有关。当3T3-L1成纤维细胞和3T3-L1脂肪细胞受到渗透休克时，两种方法测得的PtdIns-5-P水平在低渗透刺激下均显著降低。综上所述，这些结果证实PIKfyve是一种负责PtdIns-5-P生物合成的酶，并表明PtdIns-5-P在哺乳动物细胞的渗透反应途径中发挥作用。
The cellular functions, regulation and enzymology of phosphatidylinositol (PtdIns) 5-P, the newest addition to the family of phosphoinositides (PI), are still elusive. Whereas a kinase that uses PtdIns-5-P as an intracellular substrate has been assigned, a kinase that produces it remained to be identified. Here we report that PIKfyve, the enzyme found to synthesize PtdIns-5-P in vitro and PtdIns-3,5-P-2 in vitro and in vivo, is responsible for PtdIns-5-P production in a cellular context. Evidence is based on examination of two groups of cell types by two independent approaches. First, [P-32]orthophosphate-labeled cells (Sf9, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes) that show a high pressure liquid chromatography (HPLC)-detectable peak of the PtdIns-5-P head group at basal conditions demonstrated a 20-50% increase in radioactive PtdIns-5-P amounts upon expression of PIK-fyve(WT). Second, cell types (HEK293), in which the basal levels of radioactive PtdIns-5-P were undetectable by HPLC head group analysis, demonstrated higher in vitro type II PIP kinase-directed conversion of the endogenous PtdIns-5-P pool into PtdIns-4,5-P-2, when induced to express PIKfyveWT. Conversely, a decrease by 60% in the conversion of PtdIns-5-P to PtdIns-4,5-P-2 was associated with induced expression of the dominant-negative kinase-deficient PIKfyve(K1831E) mutant in HEK293 cells. When 3T3-L1 fibroblasts and 3T3-L1 adipocytes were subjected to osmotic shock, levels of PtdIns-5-P measured by both approaches were found to decrease profoundly upon a hypo-osmotic stimulus. Together, these results identify PIKfyve as an enzyme responsible for PtdIns-5-P biosynthesis and indicate a role for PtdIns-5-P in osmotic response pathways in mammalian cells.