Review
Ras trafficking, localization and compartmentalized signalling

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Abstract

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.

Highlights

► This review focussed on Ras isoform trafficking and signalling from cell surface and intracellular compartments. ► We discuss the evidence and methodologies used to investigate compartmentalized Ras signalling. ► The consequences of Ras signalling from each location are discussed in turn and we highlight areas where more work is needed.

Introduction

Ras GTPases sit near the top of signalling pathways regulating cell proliferation, differentiation and apoptosis. All cells harbour three Ras isoforms: H-Ras, K-Ras and N-Ras encoded by separate genes. Despite a high degree of sequence homology Ras isoforms are not functionally redundant. This is surprising given that the regions of the proteins that determine interactions with effectors that mediate downstream signalling, with GDP/GTP that mediates Ras activation state and with regulatory proteins that turn on and off Ras, are identical between the Ras isoforms.

Ras proteins are activated by cell surface receptors and their predominant plasma membrane localization has meant that for many years this was believed to be the exclusive site of action of Ras. More recently with the application of improved imaging and protein modification technologies intracellular pools of Ras have been identified and location-specific functions assigned. This is thought to be due to different pools and concentrations of regulators and effectors localized in each compartment that enables different types of signalling to be generated from each location. Since the main difference between Ras isoforms is their overlapping but distinctive subcellular localizations, compartmentalized signalling is believed to contribute to the lack of functional redundancy between the Ras isoforms.

Whilst the last decade has generated detailed mechanistic understanding of compartmentalized Ras signalling there is still debate over the extent to which this is important for cell function. An extreme view is that this is an experimental artifact since most work has had to rely on over-expression of tagged or mutated Ras proteins that distort the balance of normal signalling. In this review we will discuss the evidence for compartment-specific Ras signalling and how it contributes to normal Ras function.

Section snippets

Normal and mutant Ras signalling

Ras proteins are 21 kDa molecular switches that cycle between the inactive GDP-bound conformation and the active GTP-bound conformation (Fig. 1). Guanine nucleotide exchange factors (GEFs) activate Ras by catalysing the release of GDP, facilitating GTP binding due to its 10-fold higher concentration than GDP in the cytosol [1]. GTP binding induces a conformational change in the Ras switch domains (Switch 1: residues 30–40 and Switch 2: residues 60–76) revealing an effector binding site [2]. Ras

Synthesis, processing and trafficking to the plasma membrane

Ras proteins are synthesized in the cytosol and need subsequent post-translational modifications to enable them to stably associate with membranes where they can function. All three isoforms have a CAAX motif on their extreme C-termini that is sequentially processed; firstly the cysteine is isoprenylated by farnesyl protein transferase. This facilitates endoplasmic reticulum (ER) association where the AAX motif is cleaved off by Ras-converting enzyme 1 (Rce1) and finally the farnesylated

Evidence for compartment-specific Ras signalling

The challenge when studying compartment specific signalling is to separate it from the predominant signalling emanating from other locations such as the plasma membrane. Various methods are used to achieve this that mostly involve either expression of mutant or targetted Ras proteins or reporters of Ras output. An independent indicator of compartment competency to support Ras signalling is the presence of activators, effectors and co-factors for the Ras pathway. In the following section we will

Plasma membrane signalling domains

The plasma membrane is a complex, dynamic, non-equilibrium mixture of >7000 species of phospholipid, ∼30–40 mol% cholesterol and ∼25% by mass of integral and peripheral membrane proteins. Immediately underneath the lipid bilayer, which itself is asymmetric in composition, is an actin mesh that interacts with and impedes the lateral diffusion of trans-membrane proteins [80], [81], [82]. The plasma membrane is therefore not well mixed on many length and time scales, which results in non-random

Discussion

We have seen that various mechanisms can ensure signalling specificity of the Ras isoforms. Critical amongst these are their relative abundance and localized concentrations that will determine interaction efficiencies with various GEFs, GAPs and effectors over time. Importantly, the majority of strategies used to date to investigate isoform- or compartment-specific Ras signalling have abrogated the influence of these control mechanisms by overexpressing often constitutively active mutant forms

Acknowledgements

I.A.P. is a Royal Society University Research Fellow and work in his laboratory is supported by the NWCRF, BBSRC and the Wellcome Trust. Work in the laboratory of J.F.H. is supported by the NIH (GM066717).

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