REPLACEMENTS OF SINGLE BASIC-AMINO-ACIDS IN THE PLECKSTRIN HOMOLOGY DOMAIN OF PHOSPHOLIPASE C-DELTA-1 ALTER THE LIGAND-BINDING, PHOSPHOLIPASE-ACTIVITY, AND INTERACTION WITH THE PLASMA-MEMBRANE

The pleckstrin homology (PH) domain of phosphatidylinositol-specific p hospholipase C-delta 1 (PLC-delta 1) binds to both D-myo-inositol 1,4, 5-trisphosphate (Ins(1,4,5)P-s) and phosphatidylinositol 4,5-bisphosph ate (PtdIns(4,B)P-2) with high affinities. We have previously identifi ed a region rich in basic amino acids within the PH domain critical fo r ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y ., Ozaki, S., Sakuma, H., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yag isawa, H. (1994) Biochem. Biophys. Res. Commun. 205, 1563-1571). To in vestigate the role of these basic residues, we have performed site-dir ected mutagenesis replacing each of the basic amino acid in the N-term inal 60 residues of PLC-delta 1 (Lys(24), Lys(30), Lys(32), Arg(37), A rg(38), Arg(40), Lys(43), Lys(49), Arg(56), Lys(57), and Arg(60)) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for subs trate hydrolysis and membrane interactions of PLC-delta 1 were analyze d using several assay systems. Analysis of [H-3]-Ins(1,4,5)P-3 binding , measurement of the binding affinities, and measurements of phospholi pase activity using PtdIns-(4,5)P-2-containing phospholipid vesicles, demonstrated that residues Lys(30), Lys(32), Arg(37), Ar-38, Arg(40), and Lys(57) were required for these PLC-delta 1 functions; in comparis on, other mutations resulted in a moderate reduction. A subset of sele cted mutations was further analyzed for the enzyme activity toward sub strate present in cellular membranes of permeabilized cells and for in teraction with the plasma membrane after microinjection. These experim ents demonstrated that mutations affecting ligand binding and PtdIns(4 ,5)P-3 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membra ne attachment. All residues (with the exception of the K43E substituti on) found to be critical for the analyzed PLC-delta 1 functions are pr esent at the surface of the PH domain shown to contain the Ins(1,4,5)P -3 binding pocket.