Dual Activation of Phospholipase C-ϵ by Rho and Ras GTPases

Jason P Seifert,Yixing Zhou,Stephanie N Hicks,John Sondek,T Kendall Harden

doi:10.1074/jbc.m805038200

Jason P Seifert, Yixing Zhou + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m805038200

Copy DOI

Abstract

Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.

Highlights

Activation of receptors by a variety of hormones, neurotransmitters, growth factors, and other extracellular signaling molecules promotes activation of inositol lipid-specific phospholipase C (PLC)2 enzymes to catalyze hydrolysis of PtdIns[4,5]P2
Expression of PLC-⑀ with either GTPase-deficient RhoA or K-Ras resulted in PLC-⑀-dependent pull-down of the GTPase from membrane extracts of these cells (Fig. 1A)
We previously demonstrated that RhoA directly activates PLC-⑀ [33, 34], and the amount of PLC-⑀-dependent accumulation of [3H]inositol phosphates was similar with maximally effective amounts of GTPase-deficient RhoA and K-Ras or H-Ras (Fig. 1B)

Summary

Introduction

Activation of receptors by a variety of hormones, neurotransmitters, growth factors, and other extracellular signaling molecules promotes activation of inositol lipid-specific phospholipase C (PLC) enzymes to catalyze hydrolysis of PtdIns[4,5]P2. A broad range of proteins contain structural domains, including PH, FERM, ENTH, and PX domains, that bind inositol lipids necessary for their function [4, 5], and PtdIns[4,5]P2-binding proteins are implicated in a broad range of cell signaling events including membrane trafficking, changes in the actin cytoskeleton, and ion channel and transporter function [5]. PLC-␤ isozymes are activated by heterotrimeric G␣ subunits of the Gq family (6 – 8), by G␤␥ subunits (9 –11), and by the small GTPase Rac [12,13,14,15]. Rac GTPases have been shown to activate PLC-␥2, but not PLC-␥1 [19]. The sixth family member of PLC isozymes, PLC-⑀, was initially identified in Caenorhabditis elegans as a Ras-binding protein [26]. Many effectors of Ras, Rho, and Rac GTPases have been identified, knowledge of signaling proteins directly regu-

Objectives

Results

Conclusion