The Pleckstrin Homology Domains of Phospholipases C-β and -δ Confer Activation through a Common Site

Yuanjian Guo,Finly Philip,Suzanne Scarlata

doi:10.1074/jbc.m301438200

Abstract

Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.

Highlights

Mammalian inositol-specific phospholipase C (PLC)1 enzymes catalyze the hydrolysis of the signaling lipid, phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) to produce the two second messengers, 1,4,5 inositol trisphosphate, which promotes an increase in intracellular calcium, and diacylglycerol, which promotes the activation of protein kinase C
We have previously found that the emission intensity of coumarin-labeled phospholipase C-␤2 (PLC␤2) and the isolated pleckstrin homology (PH) domains of PLC␤2 and PLC␦1 increase upon membrane binding [8]
We have shown that we can transfer the activators of the catalytic core of PLC␤2 and PLC␦1 by transferring their PH domains

Summary

Introduction

Mammalian inositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of the signaling lipid, phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) to produce the two second messengers, 1,4,5 inositol trisphosphate, which promotes an increase in intracellular calcium, and diacylglycerol, which promotes the activation of protein kinase C (for review, see Refs. 1 and 2). Binding and subsequent activation of PLC␤2 by G␤␥ subunits is regulated by the N terminus of the protein which contains a pleckstrin homology (PH) domain [8, 9]. The association of the N terminus to G␤␥ subunits does not appear to be highly specific because the pleckstrin homology domain of a related protein, PLC␦, that is not activated by G proteins binds to G␤␥ with only a 4-fold weaker affinity than that of PLC␤2. These relatively close interaction energies between the two types of PLC can be compared with specific binding of the. Measurements of the membrane association of the isolated PH domains of PLC␤2 and PLC␦1 show that this region is responsible for both the affinity and specificity of membrane interaction [8]

Methods

Results

Conclusion