Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes
Hepatocyte growth factor activates phosphoinositide 3-kinase C2β in renal brush-border plasma membranes
复制标题
DOI：
10.1042/bj20020316
复制
发表时间：
2002-08-01
影响因子：
4.1
通讯作者：
Banfic, H
中科院分区：
文献类型：
--
作者：
Crljen, V;Volinia, S;Banfic, H
在肝细胞生长因子（HGF）刺激肾皮质切片后，研究了基底-外侧质膜（BLM）和刷边质膜（BBM）中肌醇脂质代谢。然而，在BLM中，1,2-二酰基甘油、PtdIns(3,4,5)P-3和PtdIns(3,4)P-2迅速增加，这表明在BLM中，HGF激活磷脂酶C （PLC）和磷酸肌肽3激酶（PI3K），而在BBM中，仅观察到HGF诱导的PtdIns3P的短暂积累，这种积累暂时延迟了BLM中的信号事件，可以被PtdIns特异性PLC抑制剂ET-18-OCH3和calpain抑制剂calpeptin阻断。表明BBM中的3-激酶激活位于BLM中PLC激活的下游，是calpain介导的事件。此外，免疫可沉淀的PI3K-C2beta活性的增加，对wortmannin （10 nM）敏感，对PtdIns比PtdIns4P作为底物有强烈的偏好，仅在HGF刺激肾皮质片的BBM中观察到，可以被Ca2+离子载体A23187模拟，并被细胞渗透的Ca2+螯合剂BAPTA-AM阻断[1,2-二-（2-氨基苯氧基）乙烷-N，N，N‘，N’-四乙酸四酰（乙酰氧基甲酯）]。在Western blots上，PI3K-C2beta在BLM和BBM中显示了180 kDa的单一免疫反应带，而在HGF刺激后，仅在BBM中出现了18 kDa的凝胶位移，这表明所观察到的酶激活是通过蛋白水解实现的。与HGF刺激后获得的BBM相比，当BBM遭受短期（15分钟）的mu-calpain暴露时，观察到类似的凝胶转移以及pi3k - c2 β活性的增加。上述凝胶转移和pi3k - c2 β活性的增加可以通过钙蛋白酶抑制剂calpeptin来阻止。本报告的数据显示，在肾细胞中，肌醇脂质信号系统在BLM和BBM之间存在空间分离，HGF主要在BLM中引起PLC和PI3K的激活，从而导致calpain介导的BBM中PI3K- c2 β的激活，并伴随PtdIns3P的增加。
Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH3 and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca2+ ionophore A23187 and blocked by the cell-penetrant Ca2+ chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2beta in BBM with a concomitant increase in PtdIns3P.