Phosphatidylinositol 4,5-Bisphosphate Binding to the Pleckstrin Homology Domain of Phospholipase C-δ1 Enhances Enzyme Activity

• DOI: 10.1074/jbc.271.41.25316
• Publication Date: 2002-07-26T15:14:15Z
• Authors: Jon W. Lomasney; Hwei-Fang Cheng; Li-Ping Wang; Y.-S. Kuan; S.-M. Liu; Stephen W. Fesik; Klim King
• Keywords: Pleckstrin homology domain; Phosphatidylinositol 4,5-bisphosphate; Homology; Phosphoinositide phospholipase C

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 co-complexed with ligands, function this remains elusive. In report, located N terminus human phospholipase C-delta1 (PLCdelta1) was found regulate enzyme activity. hydrolysis phosphatidylinositol (PI) stimulated by 4,5-bisphosphate (PIP2) dose-dependent manner EC50 = 1 microM (0.3 mol%), up 9-fold higher when 5 (1.5 mol%) PIP2 incorporated into PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 PI molar ratio PI:PS:PC 1:5:5). Stimulation specific for PIP2, since other anionic phospholipids including 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited inositol 1,4, 5-triphosphate (IP3) manner, suggesting modulatory inositol. When nested set deletions 70 amino acids from PLCdelta1 were constructed, deletion mutant enzymes all catalyzed micelle forms and activities comparable those wild type enzyme. However, effect greatly diminished more than 20 acid residues deleted terminus. To identify involved activation, functional side chains between 40 individually changed glycine. these mutations little on ability catalyze or micelles, catalytic activity mutants K24G, K30G, K32G, R38G, W36G markedly unresponsive PIP2. Analysis PIP2-stimulated dual substrate binding model catalysis revealed that micellar dissociation constant (Ks) PI/PS/PC reduced 558 53 microM, interfacial Michaelis (Km) 0.21 0.06 maximum rate (Vmax) not affected These results demonstrate major modulate Further, our as ligand suggest general mechanism regulation proteins