Replacements of Single Basic Amino Acids in the Pleckstrin Homology Domain of Phospholipase C-δ1 Alter the Ligand Binding, Phospholipase Activity, and Interaction with the Plasma Membrane

Abstract

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

Membrane Association Domains in Ca2+-dependent Activator Protein for Secretion Mediate Plasma Membrane and Dense-core Vesicle Binding Required for Ca2+-dependent Exocytosis

“…In contrast, the other mutant proteins exhibited significantly altered liposome-binding properties. The K560E mutation selectively affected interactions with PtdIns(4,5)P 2 -containing liposomes consistent with similar effects of mutation in the PLC␦1 PH domain (30). The triple mutant W537S/K538Q/R540E was deficient in binding to both PtdSer-and PtdIns(4,5)P 2 -containing liposomes.…”

“…A triple replacement mutant W537S/K538Q/R540E was generated in the ␤2 strand in a motif conserved among PH domains including PLC␦1and BTK, where cognate residues participate directly in phosphoinositide binding (29). A K560E mutation was introduced into the ␤3/␤4 loop of the CAPS PH domain, which corresponds to Lys-57 of the PLC␦1 PH domain that is important for inositol trisphosphate binding (29,30). Finally, a cluster of three basic residues in the ␤3/␤4 loop that contributes to a putative phosphoinositide binding pocket (Fig.…”

Involvement of PIP2 in RNA Polymerase I transcription

“…To further test the effect of PIP2 on transcription we compared Pol I transcription levels using nuclear extracts in which PIP2 was depleted using GSTtagged PLCd1PH domain. The PLCd1PH domain binds to the PIP2 head group with a high affinity and a single basic amino acid replacement in the N-terminal part of the domain (R40A) results in the abolishment of PIP2 binding (Yagisawa et al, 1998). When PLCd1PH domain was added to the nuclear extract, Pol I transcription was reduced significantly as compared with nuclear extract where PIP2-binding mutant PLCd1PH domain was added (Fig.…”

“…GST-tagged PLCd1PH (1-140) (pGST3) and PLCd1PH-Mut (R40A), which lacks binding to PIP2, were provided by Dr Hitoshi Yagisawa (Yagisawa et al, 1998). pECHU plasmid was constructed by PCR of the human rDNA promoter (2514 to +20) from prHU3 with EcoRI-XhoI site into pEC111/80 (Castaño et al, 2000) after the HIV LTR promoter removal.…”

“…normalized to an internal DNA control from two independent experiments (C) Nuclear extracts (NE) were depleted for PIP2 using PLCd1PH coated beads. As a control, PLCd1PH-Mut coated beads were used for depletion, since PLCd1PH-Mut fails in binding to PIP2 (Yagisawa et al, 1998). Transcription intensities were normalized by 700 bp PCR product labeled with [a-32 P].…”