Quantification of PtdIns(3,4,5)P(3) dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods.
Quantification of PtdIns(3,4,5)P(3) dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods.
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EGF 刺激的癌细胞中 PtdIns(3,4,5)P(3) 动力学的定量：PH 域介导的方法与免疫学方法的比较。
DOI：
10.1042/bj20071179
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
Backer,JonathanM
中科院分区：
文献类型：
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作者：
Yip,Shu-Chin;Eddy,RobertJ;Branch,AngieM;Pang,Huan;Wu,Haiyan;Yan,Ying;Drees,BethE;Neilsen,PaulO;Condeelis,John;Backer,JonathanM
IA类PI3Ks（磷酸肌苷3-激酶）产生二级信使PtdIns(3,4,5)P3，在许多细胞反应中起重要作用。使用GFP（绿色荧光蛋白）标记的PH （pleckstrin同源）结构域常规测量细胞膜中PtdIns(3,4,5) p3的积累。然而，通过PH结构域检测膜PtdIns(3,4,5) p3的合成和转换动力学尚未使用独立的方法进行验证。在本研究中，我们使用特异性单克隆抗PtdIns(3,4,5) p3抗体测量了表皮生长因子（EGF）刺激膜PtdIns(3,4,5) p3的产生，并将结果与ph结构域依赖方法获得的结果进行了比较。抗ptdins (3,4,5) p3染色在egf刺激的癌细胞边缘迅速积累。PtdIns(3,4,5) p3水平在1分钟时达到最高水平，并在5分钟后恢复到基础水平。相比之下，膜PtdIns(3,4,5) p3的产生，通过表位标记的dbtkph（布鲁顿酪氨酸激酶的PH结构域）的膜易位来测量，保持在大约。在4-5分钟的EGF刺激中，比基础水平高出2倍。为了确定这种差异的原因，我们通过测量LY294002处理egf刺激的细胞后PtdIns(3,4,5) p3信号的衰减来测量PtdIns(3,4,5) p3水解的速率。LY294002在处理后10秒内消除了抗PtdIns(3,4,5) p3的膜染色，表明PtdIns(3,4,5) p3在合成后几秒内就发生了转换。相比之下，一旦由EGF启动的BTKPH膜募集对LY294002相对不敏感。这些数据表明，PH结构域对PtdIns(3,4,5) p3的隔离可能会影响PtdIns(3,4,5) p3积累和周转的表观动力学；与这一假设相一致，我们发现GRP-1（磷酸肌苷一般受体1）PH结构域[与BTK一样，对PtdIns(3,4,5)P3具有特异性]在体外抑制PtdIns(3,4,5)P3的PTEN（10号染色体上缺失的磷酸酶和紧张素同源物）去磷酸化。这些数据表明，抗PtdIns(3,4,5)P3抗体是检测定位PtdIns(3,4,5)P3的有用工具，并说明了使用多种方法估计膜磷肌苷的重要性。
Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P3production using a specific monoclonal anti-PtdIns(3,4,5)P3antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3production, measured by the membrane translocation of an epitope-taggedBTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4–5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3hydrolysis by measuring the decay of the PtdIns(3,4,5)P3signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P3membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3turnover occurs within seconds of synthesis. In contrast,BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3in vitro. These data suggest that anti-PtdIns(3,4,5)P3antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.