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The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some cocomplexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-␦1 (PLC␦1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in a dose-dependent manner with an EC 50 ‫؍‬ 1 M (0.3 mol%), up to 9-fold higher when 5 M (1.5 mol%) of PIP 2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 M of PI with a molar ratio of PI:PS:PC ‫؍‬ 1:5:5). Stimulation was specific for PIP 2 , since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP 2 -mediated stimulation was, however, inhibited by inositol 1,4,5-triphosphate (IP 3 ) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLC␦1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP 2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP 2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP 2 -mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP 2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP 2 . Analysis of PIP 2 -stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (K s ) of PLC␦1 for the PI/ PS/PC vesicles was reduced from 558 M to 53 M, and the interfacial Michaelis constant (K m ) was reduced from 0.21 to 0.06 by PIP 2 . The maximum rate of PI hydrolysis (V max ) was not affected by PIP 2 . These results demonstrate that a major function of the PH domain of PLC␦1 is to modulate enzyme activity. Further, our results identify PIP 2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP 2 .

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