Elsevier

Methods in Enzymology

Volume 406, 2006, Pages 489-499
Methods in Enzymology

Quantification of Isozyme‐Specific Activation of Phospholipase C‐β2 by Rac GTPases and Phospholipase C‐ɛ by Rho GTPases in an Intact Cell Assay System

https://doi.org/10.1016/S0076-6879(06)06037-XGet rights and content

Abstract

Phospholipase C (PLC) catalyzes the hydrolysis of PtdIns(4,5)P2, which results in both formation of the second messengers Ins(1,4,5)P3 and diacylglycerol and alteration in the membrane association and/or activity of PtdIns(4,5)P2‐binding proteins. The existence of 13 different PLC isozymes suggests multiple mechanisms of regulation of inositol lipid signaling, and the recent realization that Rho‐family GTPases directly bind and activate certain PLC isozymes has added to this potential diversity of inositol lipid–related signal transduction. With the goal of delineating a less labor‐intensive method for quantification of intracellular inositol phosphate production, we have applied a commercially available yttrium silicate RNA binding resin selective for inositol phosphates to develop a high‐throughput inositol phosphate scintillation proximity assay (SPA). We highlight the utility of this assay using COS‐7 cells robotically transfected in a 96‐well format. This method is readily applied to quantify activation of PLC by receptors and G proteins, and we illustrate here the selective activation of PLC‐β2 by Rac but not by Rho GTPases and the selective activation of PLC‐ɛ by Rho but not Rac GTPases.

Introduction

The central role of phospholipase C (PLC)–catalyzed breakdown of membrane phosphoinositides in the Ca2+‐mobilizing and protein‐kinase C–activating action of a broad range of growth factors, hormones, neurotransmitters, and other extracellular stimuli was established in the 1980s (Berridge, 1987). A large family of PLC isozymes subsequently was identified, and this group of cell signaling proteins now includes PLC‐β, ‐γ, ‐δ, ‐ɛ, ‐ζ, and ‐η isozymes (Rhee, 2001). All PLC‐β isoforms are directly activated by Gα‐subunits of the Gq family of heterotrimeric G proteins, and PLC‐β2 and PLC‐β3 additionally are regulated by Gβγ‐subunits. In contrast, PLC‐γ1 and PLC‐γ2 contain SH2 and SH3 domains and are regulated by receptor and nonreceptor tyrosine kinases.

Although early work from Wakelam et al. (1986) as well as Cantley and coworkers (Fleischman et al., 1986) suggested that Ras regulates inositol lipid hydrolysis, working models of hormonal regulation of phospholipase C–mediated cell signaling principally have included Gαq‐ and Gβγ‐mediated regulation of PLC‐β isozymes and tyrosine kinase‐regulated PLC‐γ isozymes. This long‐held view has proven to be an oversimplification, and the groundbreaking work of Gierschik and coworkers illustrating activation of PLC‐β2 in neutrophils by Cdc42 and Rac presaged an increased appreciation of regulation of inositol lipid metabolism by Ras superfamily GTPases (Illenberger 1998, Illenberger 2002). This idea has been augmented by the recent discovery of PLC‐ɛ as a Ras binding protein (Kelley 2001, Lopez 2001, Song 2001).

Not only is PLC‐β2 directly activated by Gαq and Gβγ, but it also is directly activated by GTP‐dependent binding of Rac1, Rac2, Rac3, and (to a lesser extent) Cdc42 (Illenberger et al., 2002). Interaction with the amino terminal PH domain entirely accounts for Rac binding to PLC‐β2, as we initially illustrated by surface plasmon resonance (Snyder et al., 2003) and have more recently unambiguously confirmed in the crystal structure of a complex of GTP‐Rac1 and PLC‐β2 (Jezyk, M. et al., unpublished observations).

The work of Kataoka (Song et al., 2001) and Kelley, Smrcka and colleagues (Kelley et al., 2001) established that Ras and Rap bind in a GTP‐dependent manner to the RA domains present in the carboxy terminus of PLC‐ɛ. Interestingly, PLC‐ɛ also contains an amino terminal Cdc25 GEF domain and is an upstream regulator of Ras, Rap, and potentially other GTPases (Jin 2001, Lopez 2001). Although PLC‐ɛ is not activated by Gαq, overexpression of Gα12 or Gα13 results in activation of this isozyme in cell‐based assays of inositol lipid hydrolysis (Lopez 2001, Wing 2001). The fact that Gα12/13 activates RhoGEFs suggested to us that Rho also might be a direct regulator of PLC‐ɛ. Indeed, RhoA, RhoB, and RhoC all activate PLC‐ɛin vivo, and this activity is independent of the Ras‐binding RA domains in the PLC‐ɛ carboxy terminus (Wing et al., 2003). Our recent work with purified proteins has confirmed that the activation of PLC‐ɛ by Rho GTPases is direct and occurs through interaction in the catalytic core of the isozyme (Seifert et al., 2004).

The discovery that PLC‐β2 and PLC‐ɛ are effectors of Rho GTPases has made necessary the facile evaluation of the capacity of multiple Ras superfamily GTPases and mutants thereof to regulate inositol lipid hydrolysis in an intact cell context. The capacity of heterotrimeric G protein–coupled and tyrosine kinase receptors to stimulate PLC has been evaluated over the past two decades by preincorporation of [3H]inositol into phosphoinositide pools, incubation of cells or tissues in the presence of hormones and LiCl, and isolation of [3H]inositol phosphates by Dowex chromatography (Berridge et al., 1982). We recently have developed a unified system that applies robotic transfection of COS‐7 cells with expression vectors for Ras GTPases and PLC isozymes in a 96‐well format, followed by quantification of [3H]inositol phosphates using scintillation proximity assay (SPA) beads that circumvents laborious separation of [3H]inositol phosphates using ion exchange columns. The methods underpinning this utility are described here as is the application of these techniques to illustrate the selective activation of PLC‐β2 by Rac GTPases and PLC‐ɛ by Rho GTPases.

Section snippets

Cell Culture

COS‐7 cells are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 4 mMl‐glutamine, 200 U penicillin, and 0.2 mg/ml streptomycin (all obtained from Gibco, Invitrogen Corp.) at 37° in a 10% CO2/90% air‐humidified atmosphere. COS‐7 cells are maintained during routine passage in culture in 162‐cm2 (T‐162) tissue culture flasks (Corning Costar). A confluent T‐162 flask of COS‐7 cells yields approximately 1 × 107 cells. To plate cells for assay in 96‐well

Concluding Remarks

The existence of a broad family of at least 13 different mammalian PLC isozymes suggests multiple physiological roles of these signaling proteins, and PLC isozymes are important in many ways that transcend their historically considered function in hormone‐regulated Ca2+ signaling. These enzymes process multiple forms of upstream regulation and coordinate multiple downstream responses unrelated to those initially associated with PLC action. The realization that Rho superfamily GTPases

Acknowledgments

This work was supported by National Institutes of Health grants GM29536, GM57391, and GM65533. D. B. recognizes support from the Pharmaceutical Research and Manufacturers of America Foundation. J. S. recognizes support from the Pew Charitable Trusts.

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