Studies of inositol lipid-specific phospholipase C (PLC) have elucidated the main regulatory pathways for PLCbeta and PLCgamma but the regulation of PLCdelta isoenzymes still remains obscure. Here we demonstrate that an increase in Ca2+ ion concentration within the physiological range (0.1-10 microM) is sufficient to stimulate PLCdelta1, but not PLCgamma1 and PLCbeta1, to hydrolyse cellular inositol lipids present in permeabilized cells. The activity of PLCdelta1 is further enhanced in the presence of phosphatidylinositol transfer protein (PI-TP). Both full activation by Ca2+ ions and stimulation in the presence of PI-TP require an intact PH domain involved in the membrane attachment of PLCdelta1. The physiological implication of this study is that PLCdelta1 could correspond to a previously uncharacterized PLC responsible for Ca2+ ion-stimulated inositol lipid hydrolysis observed in many cellular systems.
The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-␦1 (PLC-␦1) binds to both D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, The pleckstrin homology (PH) 1 domain has been initially identified as a region of sequence similarity of about 120 amino acid residues (3, 4). At the last count, more than 100 proteins have been reported to have this sequence motif; many of these proteins are involved in cellular signaling and cytoskeletal functions (5-8). Studies of several PH domains using x-ray crystallography or NMR (9 -12) revealed a conserved structural module containing a seven-stranded -sandwich formed by two orthogonal antiparallel -sheets and a C-terminal amphiphilic ␣-helix. The loops between the -strands, particularly the 1/ 2, 3/4, and 6/7, differ greatly in length and sequence. Each PH domain is electrostatically polarized, and the most variable loops coincide with the positively charged face.By analogy with other conserved structural modules (e.g. SH2 and SH3 domains), it has been proposed that the PH domain could be involved in signaling by mediating intermolecular interactions. Consequently, a great effort has been made to identify ligand(s) for this domain. Although there are examples of PH domains involved in protein-protein interactions (e.g. binding of G␥ by -adrenergic receptor kinase PH domain (13) or recognition of phosphotyrosine by Sch PH/PTB domain (14)) there is an increasing evidence that many PH domains interact with different inositol lipids and inositol phosphates (15,16). In this respect, the PH domain of phospholipase C-␦1 (PLC-␦1) has been studied most extensively. Determination of association constants for different inositol lipids and their head groups (1, 2, 17), and relative abundance of these phospholipids in the cell identified PtdIns(4,5)P 2 as a potentially important physiological ligand (18,19). Ins(1,4,5)P 3 can bind to the same binding pocket as the head group of
p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-∆ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1p130). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1p130 cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1p130 cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1p130. This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.
The pleckstrin homology (PH) domains of phospholipase C (PLC)-d1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] by PLC-d1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca 2+ -dependent activation of PLC-d1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-d1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P 2 is regulated by changes in free Ca 2+ concentration within the physiological range. A Ca 2+ dependent increase in the binding to PtdIns(4,5)P 2 was observed with a full-length PLC-d1, while the isolated PH domain did not show any Ca 2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca 2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-d1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca 2+ dependent increase in the binding to PtdIns(4,5)P 2 . The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P 2 showed no Ca 2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-d1 EF-hand motifs, the Ca 2+ dependent binding was again observed. These results suggest that the binding of Ca 2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P 2 mediated by the PH domain.Keywords: pleckstrin homology domain; EF-hand motif; calcium; phosphoinositide; inositol trisphosphate; phospholipase C-d.A number of modular domains have been recognized as mediators of distinct intermolecular interactions central to a variety of cellular processes and, in particular, cellular signalling. One such domain, the pleckstrin homology (PH) domain, was originally identified in pleckstrin and has subsequently been found in more than 100 proteins with diverse cellular functions. The PH domain has been the focus of extensive structural and functional studies, which revealed a common fold for this domain and also identified a number of potential ligands [1±4]. Although there are examples of PH domains being involved in protein±protein interactions (e.g. interactions with Gbg, protein kinase C, phosphotyrosine residues, an acidic protein motif or myosin II [5±13]), there is a growing body of evidence that many PH domains interact with different inositol lipids and inositol phosphates [1±4,14,15].Members of phosphoinositide-specific phospholipase C (PLC) b, g and d families that play a key role in production of second messenger molecules after stimulation by many extracellular agonists, have an N-terminal PH domain. In these molecules, the PH domain is one of four conserved domains (the PH-, EF hand-, catalytic-and C2-domain) comprisi...
p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.
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