Phosphatidyunositol-4,5-bisphosphate (pip₂) binding to the pleckstrin homology (PH) domain of phospholipase C (PLC) δ1 enhances enzyme activity

The PH domain is a newly recognized protein module believed to play an important role in signal transduction. Using deletion and point mutagenesis we have determined the function of the PH domain located in the N-terminus of human PLC δ1. A nested set of PH domain deletion mutants were constructed and found to catalyse the hydrolysis of the micelle forms of phosphatidylinositol (PI) and PIP₂ with specific activities comparable to native enzyme. However, deletion of 20 amino acids or more results in defective high affinity binding of PIP₂. The functional consequences of high affinity PIP₂ binding was elucidated by examining the effect of PIP₂ on the ability of the enzyme to catalyse the hydrolysis of ³H-PI in defined phospholipid (mole fraction; PI:PC:PS = 1:5:5) vesicles. In this PI-vesicle hydrolysis assay system, the activity of native PLC δ1 is stimulated (up to 10-fold) in a dose dependent manner by PIP₂. In contrast, PIP₂ scarcely enhanced the catalytic activity of the truncated enzymes. Specific residues involved in high affinity PIP₂ binding and enzyme activiation were determined by mutating individual amino acids with functional side chains between residues 20 to 40 to glycine. While all these mutants catalyse the hydrolysis of PI or PIP₂ micelles, mutants K24G, K30G, K32G, R38G or W36G have impaired high affinity PIP₂ binding, and impaired PIP₂ stimulation of catalysis. This identifies PIP₂ as a true ligand for PH domains, and demonstrates that a major function of PH domains is to modulate enzyme activity. Further, our results identify a novel mechanism for the activation of phospholipase C, and suggest a general mechanism for the regulation of other proteins by PIP₂.