Related Experiment Video

Updated: Jul 30, 2026

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
07:33

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Published on: April 26, 2011

Phospholipase C(epsilon): a novel Ras effector

G G Kelley1, S E Reks, J M Ondrako

  • 1Department of Medicine, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA. kelleyg@mail.upstate.edu

The EMBO Journal
|February 17, 2001
PubMed

Insights

Researchers discovered a new class of enzyme, phospholipase C-epsilon (PLC-epsilon), directly regulated by Ras proteins. This finding links Ras signaling pathways to PLC signaling, revealing a novel biological mechanism.

Area of Science:

  • Biochemistry
  • Cell Signaling
  • Molecular Biology

Background:

  • Mammalian phosphoinositide-specific phospholipase C (PLC) enzymes are crucial for cellular signaling.
  • Three known classes (PLCbeta, PLCgamma, PLCdelta) are regulated by G-proteins, tyrosine kinases, and calcium.
  • The precise mechanisms of PLC regulation are not fully understood.

Purpose of the Study:

  • To identify and characterize a novel class of mammalian phosphoinositide-specific phospholipase C.
  • To elucidate the regulatory mechanisms of this newly discovered PLC class, particularly its interaction with Ras proteins.
  • To understand how this new PLC class integrates Ras signaling with other cellular pathways.

Main Methods:

  • Protein purification and characterization of recombinant PLC-epsilon.
  • Analysis of domain structure, including GTP exchange factor (GRF CDC25) and Ras-binding (RA) domains.
  • In vitro binding assays to assess interactions with H-Ras and GTP-dependent binding.
  • Cell-based assays (COS-7 cells) to study activation by Ras mutants and domain deletion effects.
  • Site-directed mutagenesis to investigate Ras-effector interactions.

Main Results:

  • Identification of a fourth mammalian PLC class, designated PLC-epsilon.
  • PLC-epsilon possesses conserved PLC domains, a GRF CDC25 domain, and two RA domains (RA1 and RA2).
  • The RA2 domain binds H-Ras in a GTP-dependent manner, while RA1 exhibits low-affinity, GTP-independent binding.
  • Constitutively active H-Ras (Q61L) stimulates PLC-epsilon activity in co-expressed cells, dependent on both RA1 and RA2 domains.
  • Mutational analysis confirms a conserved Ras-effector interaction and reveals specific activation profiles by Ras effector domain mutants.

Conclusions:

  • PLC-epsilon represents a novel fourth class of mammalian phosphoinositide-specific phospholipase C.
  • This enzyme class is directly regulated by Ras proteins, establishing a direct link between Ras signaling and PLC activity.
  • PLC-epsilon acts as a molecular bridge, integrating the Ras signaling pathway with critical phosphoinositide hydrolysis pathways.

More Related Videos

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes08:49

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Published on: March 14, 2021

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA210:31

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2

Published on: September 26, 2025

Related Concept Videos

Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...