PX-FERM proteins

Abstract

Endosomes are the primary organelle where decisions are made as to whether endocytosed proteins will be sorted into degradative trafficking pathways or recycled back to the plasma membrane. This balance between cellular uptake and recycling regulates the plasma membrane composition and is therefore critical for many cellular processes such as nutrient uptake, neuronal transmission and cell migration.¹ In addition to its well-known role in membrane trafficking, the endosome is increasingly being recognized as a critical cellular domain for regulated cell signaling. We recently showed that several proteins that regulate endosomal recycling, SNX17, SNX27 and SNX31 are structurally and functionally related.² These proteins use an unusual FERM domain to bind specific endosomal cargo molecules, and most interestingly, we also found that these proteins use the same FERM domain to associate with the activated Ras small GTPase. Here we speculate on the potential dual role of the PX-FERM proteins in endosomal transport and as scaffolds that may be involved in endosomal Ras signaling processes.

The communication of a cell with its environment is controlled by a symphony of molecular interactions, membrane trafficking events and cell signaling responses to external stimuli. The composition of the plasma membrane in terms of the receptors, adhesion molecules and channels that are present at the cell surface, is regulated by the net balance of their exocytosis, endocytosis, degradation and recycling. A key organelle involved in maintaining this balance is the endosome, actually a heterogeneous population of membrane-bound compartments evolving from early endosomes, to late endosomes that deposit material in the lysosome or recycling endosomes for retrieval of material back to the cell exterior. In the context of cell signaling, transport of material through the endosomal compartments can regulate signal output by the relatively obvious mechanism of controlling the level of ligand-activated signaling receptors at the cell surface; but it is also now apparent that endosomes can function as platforms for differential assembly of signaling complexes leading to distinct signaling outcomes.^3–6

The regulation of endosomal trafficking is achieved by the endowment of compartment specific proteins such as the Rab GTPases, the ESCRT machineries and various SNAREs and membrane tethers, and lipids such as the phosphoinositides PI(3)P and PI(3,5)P₂. The Phox-homology (PX) domain containing proteins (often referred to as sorting nexins (SNXs)) are a functionally and structurally diverse family of peripheral membrane molecules that are engaged in various endocytic and protein trafficking processes. PX proteins are also fast emerging as vital molecules in signal transduction pathways. A distinct attribute of the PX domain is its ability to associate with phosphoinositides to mediate the localization of PX proteins to various subcellular membranous compartments.^7,8 A number of studies have shown that most PX proteins have a preferential affinity for binding PI(3)P, which drives their predominantly early endosomal localization. The molecules belonging to the PX protein family typically possess additional functional modules such as Bin/amphiphysin/Rvs (BAR), Src-homology 3 (SH3), regulator of G-protein signaling (RGS) and PSD-95/discs large/zona occludens (PDZ) domains.^9,10 Thus many PX proteins are predicted to function as scaffolds for the appropriate spatio-temporal assembly of membrane trafficking and signaling complexes.

SNX17, SNX27 and SNX31 PX Proteins Show Ras-Binding Activity

Recently, we defined a novel sub-family of PX proteins SNX17, SNX27 and SNX31 that in addition to the canonical PX domain also contain a structurally distinct band4.1/ezrin/radixin/moesin (FERM) domain at their C-terminus.² This unusual FERM domain is composed of the canonical F1 and F3 sub-domains found in all FERM proteins, structurally related to ubiquitin and phosphotyrosine binding (PTB) domains respectively, however, they possess an altered central F2 helical sub-domain. In addition SNX27 is unique within the PX family in possessing an N-terminal PDZ domain.

A number of studies have established that SNX17 regulates endosomal trafficking of receptors containing the sorting peptide motif Asn-Pro-Xaa-Tyr (NPxY), in particular regulating recycling of the amyloid precursor protein (APP) and members of the lipoprotein receptor family such as low-density lipoprotein receptor (LDLR).^11–13 Independently SNX27 has been shown to regulate endosomal recycling via binding of its N-terminal PDZ domain to receptors such as potassium channels, N-methyl-D-aspartate receptors and adrenoreceptors that contain C-terminal PDZ domain binding motifs (PDZbm).^14–16 The discovery via bioinformatic and structural analyses that SNX17 and SNX27 (and the close SNX17 homolog SNX31) each contain a similar FERM domain subsequently allowed us to show that SNX27, like SNX17 is also able to bind receptors with NPxY motifs.²

Previous reports indicated SNX27 has a Ras-association (RA) domain, raising the possibility that it might be an endosomal Ras-effector protein.^9,10 However, our new data demonstrates that the SNX27 “RA domain” is not a stand-alone structure as seen in all other RA domain proteins. It is in fact a sub-domain (F1) of the FERM module, the confusion arising due to the shared structural similarity and limited sequence homology of RA and F1 sub-domains with ubiquitin. Nonetheless, this raised the intriguing possibility that the FERM F1 module of SNX27 could have Ras-binding activity, and that this activity may be shared with SNX17 and SNX31. Subsequently we were able to confirm using in vitro purified proteins that the PX-FERM molecules all show specific association with activated GTP-bound H-Ras.² While it remains to be demonstrated that the PX-FERM proteins directly regulate Ras in vivo, we take this opportunity to speculate on this interaction and the potential role of PX-FERM (and PDZ-PX-FERM) proteins as scaffolds for coordinating both trafficking and signaling complexes.

The FERM Domain as a Novel Ras Interaction Module

H-Ras, N-Ras and K-Ras are almost identical within their GTPase effector-binding domains, and highly related to other Ras-family members.^17,18 Thus while our experiments demonstrate association with GTP-bound H-Ras, it is not yet possible to link PX-FERM proteins to a specific Ras protein functionally. It will require further experiments to gain a deeper understanding of the functional interactions of specific Ras and PX molecules. This unveiling of the small GTPase binding capability of PX-FERM proteins however, places them as unique FERM domain containing proteins with potential to couple endosomal sorting and cell signaling pathways. More generally, the recognition that the FERM domain of PX proteins has functional Ras-binding activity leads to the prediction that other FERM domain proteins outside of the PX-FERM family may have similar binding activity. This possibility is supported by the identification of Krit1 as an effector of the Ras family protein Rap1, interacting with the Rap1 GTPase via its FERM domain to stabilize junctional integrity in epithelial cells.^19–22 Demonstration of a direct relationship between Ras GTPases and FERM domain-containing protein families will have a major impact on many different fields of biological research, given the central roles of both Ras GTPases and FERM domain proteins (such as Janus kinases or Talin) in diverse signal transduction pathways that have thus far been considered as distinct from each other.

Ras Trafficking at Endosomes

The Ras small GTPase family comprises 36 members in the human genome, that act as signaling nodes where a stimulus directs guanine nucleotide exchange factors (GEFs) to activate the Ras molecules, and in turn the Ras proteins direct a broad array of signals through their association with downstream effectors such as protein and lipid kinases.^17,18 Central to the spatiotemporal signaling cascades managed by Ras GTPases is the localization of these molecules, which is primarily controlled by the C-terminal hypervariable regions (HVR) and CAAX motifs. Post-translational modification of the CAAX motifs by prenylation, proteolysis and palmitoylation controls the recruitment of the Ras proteins to intracellular membranes. In addition, vesicular transport of palmitoylated Ras proteins controls aspects of their sub-cellular localization including endocytosis within clathrin-coated vesicles.^23–27 Transport through recycling endosomes to the cell surface has recently been shown for palmitoylated H and N-Ras,²⁶ and previously it was found that the Ras-related GTPase Rap1 is recruited from endosomes to the cell surface upon cell stimulation by mitogens.²⁸ Interestingly, this recruitment was blocked by the general transport inhibitor N-ethylmaleimide, or by specific disruption of the recycling endosome by expression of the dominant negative Rab11 protein. The fact that SNX17 and SNX27 are observed in early and recycling endosomes and have a demonstrated role in endosomal recycling, suggests the tantalizing possibility that PX-FERM proteins could act as vesicular coats or adaptors for Ras trafficking to the cell surface.^13,29,30

Ras signaling at Endosomes

Cells internalize various receptor-ligand complexes to maintain homeostasis and regulate signaling propagation thereof. Perhaps not surprisingly therefore, in the last decade or so endosomes themselves have been firmly established as sites for propagation of cell signaling.^3–5,31 More specifically, recent assessment of the spatiotemporal aspects of Ras signaling has demonstrated the existence and importance of signaling events managed by Ras on intracellular membranes including endosomes, post internalization.^23,24,32 A number of studies such as those of Lu and colleagues (2009),²⁵ Jiang and Sorkin (2002),³³ or Roy et al. (2002) have demonstrated the presence of several activated Ras isoforms on endomembranes. However, the exact nature of Ras-mediated signaling from endosomes, whether this is distinct from signaling from the plasma membrane or other compartments, and the identities of endosome-specific Ras effectors and regulators all still remain poorly defined.

PX-FERM Proteins as Scaffolds or Adaptors in Signaling and Trafficking

It is a well-established principle that cells control signaling specificity by organizing signaling molecules into discrete assemblies, and by coupling such assemblies to specific cellular compartments.^35–38 The formation of signaling assemblies is achieved through the action of scaffolds or adaptor/docking proteins, modular proteins with multiple binding domains that act to tether the signaling components together. For example, growth factor-stimulated Ras signal propagation through the MAP kinase pathway is coordinated by the coupling of MAP kinases by the scaffold protein kinase suppressor of Ras-1 (KSR). Upstream of kinase activation stimulated growth factor receptors recruit adaptor proteins via SH2 and PTB domain interactions. Such adaptors clearly share much in common with the PX-FERM proteins, generally possessing a membrane binding module such as a PH or FYVE domain, and other protein-protein interaction modules for recruiting effectors or regulatory molecules.

While still highly speculative, we propose that a potential function of the PX-FERM proteins may be to act as endosome-specific scaffolds/adaptors with a role in intracellular signal propagation mediated by a Ras-related protein(s). Given the proven role of PX-FERM proteins in transport this may also be coupled to the trafficking of signaling receptors and/or Ras itself. Key to such a proposal is the demonstrated ability of the PX-FERM proteins to function as modular recruitment platforms, binding transmembrane receptors from diverse families, as well as cytoplasmic proteins including cytohesin associated scaffolding protein (CASP),³⁹ Src family kinases,⁴⁰ lipid kinases²⁹ and regulators of cytoskeletal function.^15,41 There is some preliminary evidence that PX-FERM proteins can impact on Ras-mediated signal transduction. In T cells depleted of SNX27 by siRNA, there is a marked increase in the level of phosphorylated ERK stimulated by contact with antigen presenting cells, implying that SNX27 can suppress normal MAP kinase signal cascades to some extent.⁴² It should also be noted that the PX-FERM proteins will likely play a distinct role in signal transduction by controlling the trafficking of signaling receptors themselves. A clear example is the transport of the β2-adrenergic G-protein coupled receptor (GPCR) by SNX27,³⁰ and it is also highly probable that tyrosone kinase receptors containing NPxY sorting signals will be regulated at the endosome by the PX-FERM family.

Future Directions

We propose a model in Figure 1 for the endosomal association of PX-FERM proteins with cargo molecules, Ras and potential effectors. In this model PX-FERM proteins are recruited to endosomes via the lipid PI(3)P, and here bind to cargo receptors containing either PDZ binding motifs (by SNX27) or NPxY signals (by all PX-FERM molecules). The FERM domain is then able to couple to a Ras-related GTPase, and we predict this interaction should not be mutually exclusive with receptor association. While there is some evidence for a negative role in specialized Ras-mediated signaling processes,⁴² this is still preliminary and requires further confirmation and investigation of more general systems. Finally the PX-FERM proteins regulate receptor cargo recycling within retromer-coated tubules,³⁰ and it is possible that Rastrafficking will be coordinated by the same pathways.

Figure 1.

Speculative model for the membrane-coupled interaction of PX-FERM proteins with ras GTPases and transmembrane cargo molecules transiting through the endosome for recycling to the cell surface. The PX-FERM proteins dock to the periphery of early endosomal limiting membranes by associating with PI(3)P. Here they interact with cargo receptors containing the NPxY sequences via the F3 module of the FErM domain. SNX27 has an additional PDZ domain (indicated by dashed border) that engages numerous cargo molecules containing PDZ binding motifs. In addition, PX-FERM proteins may interact with a range of cytosolic effectors such as Kif1b. At the endosomal membrane PX-FERM proteins are primed to bind to activated ras GTPases where they may act as a scaffold/adaptor for the assembly of signaling complexes. There are however, many questions that remain to be answered, and indicated on the diagram are three questions we feel of particular importance. (1) What is the mode of PX-FERM interaction and which ras family molecules are specifically targeted? (2) what is the impact of PX-FERM protein interaction with small Ras GTPases on signaling? Does this interaction facilitate signal attenuation or propogation? (3) Alternatively, or in addition to a role in signaling, do PX-FERM proteins act as adaptors that modulate vesicular trafficking of Ras GTPases?

There are clearly many questions remaining. Of primary importance is to confirm the nature of Ras-binding by the PX-FERM proteins, and most importantly the specificity of this interaction in terms of which Ras family molecules are targeted. Until the Ras binding partner(s) are identified it will be difficult to probe their functional association with the PX-FERM proteins. That endosomes are important sites of cell signal propagation is no longer considered controversial, but it is still unknown if they define spatial platforms for distinct signal outputs or merely act to extend signal processes initiated at the cell surface. Once the role of PX-FERM proteins in Ras signaling is firmly established, the foundation will be set to delineate important pathways and mechanisms that link both endosomal signal transduction and membrane trafficking.

Extra View to: Ghai R, Mobli M, Norwood SJ, Bugarcic A, Teasdale RD, King GF, et al. Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases. Proc Natl Acad Sci USA. 2011;108:7763–7768. doi: 10.1073/pnas.1017110108.