title: Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.
abstract: 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-OCH(3) 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-C2 beta 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 Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta 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-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta 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-C2 beta in BBM with a concomitant increase in PtdIns3P.
authors.affiliation: Department of Physiology and Croatian Institute for Brain Research, School of Medicine, University of Zagreb, Salata 3, 10000 Zagreb, Croatia.
authors.forename: Vladiana
authors.initials: V
authors.lastname: Crljen
authors.forename: Stefano
authors.initials: S
authors.lastname: Volinia
authors.forename: Hrvoje
authors.initials: H
authors.lastname: Banfic
mesh_terms: D000818:Animals; D002154:Calpain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D019852:Diacylglycerol Kinase / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D004075:Diglycerides; D007527:Isoenzymes; D010716:Phosphatidylinositols; D017228:Hepatocyte Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D019852:Diacylglycerol Kinase; D002154:Calpain