Eps8 in the midst of GTPases

Eps8 in the midst of GTPases

The International Journal of Biochemistry & Cell Biology 34 (2002) 1178–1183 Molecules in focus Eps8 in the midst of GTPases Pier Paolo Di Fiore a,b...

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The International Journal of Biochemistry & Cell Biology 34 (2002) 1178–1183

Molecules in focus

Eps8 in the midst of GTPases Pier Paolo Di Fiore a,b,c,∗ , Giorgio Scita a a

Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti, 435, 20141 Milan, Italy b IFOM, The FIRC Institute for Molecular Oncology, 20134 Milan, Italy c Dipartimento di Medicina Chirurgia ed Odontoiatria, Universita’ degli Studi di Milano, 20122 Milan, Italy Received 12 March 2002; received in revised form 23 April 2002; accepted 23 April 2002

Abstract Eps8, originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR), displays a domain organization typical of a signaling molecule that includes a putative N-terminal PTB domain, a central SH3 domain, and a C-terminal “effector region”. This latter region directs Eps8 localization within the cell and is sufficient to activate the GTPase, Rac, leading to actin cytoskeletal remodeling. Eps8 binds, through its SH3 domain, to either Abi1 (also called E3b1) or RN-tre. Abi1 scaffolds together Eps8 and Sos1, a dual specificity guanine nucleotide exchange factor for Ras and Rac proteins, thus facilitating the formation of a trimeric complex, in turn required for activation of Rac. On the other hand, RN-tre, a Rab5 GTPase activating protein, by entering in a complex with Eps8, inhibits EGFR internalization. Furthermore, RN-tre competes with Abi1 for binding to Eps8, diverting the latter from its Rac-activating function. Thus, depending on its engagement in different complexes, Eps8 participates to EGFR signaling through Rac and endocytosis through Rab5. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Eps8; EGFR; Rac; Rab5

1. Introduction Eps8 (an acronym for EGFR pathway substrate #8) was originally isolated by an expression cloning strategy, designed to isolate intracellular substrates for the kinase activity of the EGFR [3]. It is a protein of 97 KDa, which is efficiently tyrosine phosphorylated Abbreviations: Eps8, EGF receptor pathway substrate 8; EGFR, Epidermal growth factor receptor; PTB, phosphotyrosine binding domain; SH3, Src homology domain 3; RTK, receptor tyrosine kinases; SAM-PNT, sterile alpha-pointed domain; PI3K, phosphatidylinositol 3 kinase ∗ Corresponding author. Tel.: +39-0257-489855; fax: +39-0257-489851. E-mail addresses: [email protected] (P.P. Di Fiore), [email protected] (G. Scita).

by a variety of tyrosine kinases, both of the receptor (RTKs) and non-receptor type [3]. Eps8 is ubiquitously expressed, and maps to human chromosome 12p13.2 [7]. Eps8 homologues are found in Drosophila and in the nematode C. elegans [21], thus underscoring an important role conserved in evolution. In mammals, at least three other Eps8-related genes are present, which display collinear topology and high degree of similarity to Eps8 ([15] and our unpublished observations). Initial experiments indicated how the overexpression of Eps8 conferred increased mitogenic responsiveness to EGF and increased the transforming ability of EGFR [13], suggesting its involvement in RTK-activated pathways leading to cell proliferation [3].

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2. Synthesis and degradation

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3. Structure

The PTB domain is a protein–protein interaction module, which, despite its name, binds to a variety of peptides both in a phosphotyrosine-dependent and -independent fashion. Binding of PTBs to phospholipids has been demonstrated as well. This broad range of interactions, together with the observation that PTB domains are found in a variety of proteins involved in diverse cellular processes, indicates that PTBs can exert different functions ranging from receptor signaling to protein targeting (see [4] for review). No binding partner for the PTB domain of Eps8 is yet known. SAM-PNT domains belong to a subfamily of the SAM domains known to mediate homo- and hetero-oligomerization and specific protein–protein interaction [19]. It was originally identified in the ETS1 family of transcription factor and shown to mediate the interaction with the transcriptional repressor, DAXX [11]. The prevalent cytoplasmic localization of Eps8 makes it unlikely that a similar function is exerted by its SAM-PNT domain, suggesting rather that it may contribute to other protein–protein interactions (see later).

3.1. General topology

3.2. The SH3 domain of Eps8

Computer-assisted analysis of the predicted amino acid sequence of Eps8 (Fig. 1) reveals a structural organization typical of a signaling molecule, containing (from N to C terminus) a phosphotyrosine binding protein (PTB) domain, a SH3 domain and a sterile alpha-pointed (SAM-PNT) domain. It should be pointed out, that only the central SH3 domain of Eps8 has been extensively characterized both from a structural and a biological point of view and it will be discussed further later.

A canonical SH3 fold consists of two anti-parallel beta sheets packed against each other at an approximate right angle [14]. The crystal structure of the SH3 domain of Eps8 revealed an intertwined dimer, characterized by “strand exchange”, in which the two anti-parallel beta sheets are contributed by different polypeptide chains. Surprisingly, this results in half-dimers whose folds are superimposable to that of a canonical SH3 module. An important consequence of the dimeric configuration is that the proline

To date, no information is available on the synthesis and degradation of Eps8. Eps8 undergoes tyrosine phosphorylation, which is triggered by activation of RTK. In the model system of NIH3T3 cells overexpressing EGFR, receptor-saturating doses of EGF induce tyrosine phosphorylation of about 30% of the total Eps8 pool [13]. In comparison, two other known EGFR substrates, Ezrin and PLC␥ showed tyrosine phosphorylation of 10 and 3% of the total pool, respectively. Additionally, constitutive tyrosine phosphorylation of Eps8 is specifically detected in a variety of tumor cells [13], pointing to a role of this post-translational modification in cell proliferation. The identification of the tyrosine phosphorylation site(s) of Eps8 will be instrumental to validate its potential functional role and to elucidate the molecular mechanisms of its action.

Fig. 1. Domain organization of Eps8. Human Eps8 is 821 amino acids long. The domains, predicted by analysis of the primary sequence, are indicated: PTB, (aa: 60–197); SAM-PNT, C-terminal sterile alpha, pointed domain (aa: 709–783). The SH3 domain (aa: 535–586) mediates the interaction with Abi1 and RN-tre. The cross-hatched bar indicates the EGFR binding region (aa: 296–362). The black bar indicates the “effector region” (aa: 648–821) responsible for the interaction with Sos1 and F-actin.

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helix-binding groove is partially occluded in the context of the hybrid-dimer, impeding binding to ligands [9]. More recently, a monomeric structure of the SH3 domain of Eps8 has been reported, as obtained from crystals grown at low pH; in this case, the SH3 domain of Eps8 displayed a conserved SH3 fold [8]. Whether the bona fide configuration of the SH3 of Eps8 in vivo is monomeric or dimeric is not known. It was shown, however, that the isolated Eps8–SH3 exists in vitro as dimeric and monomeric forms in equilibrium [9], and that ligand binding occurs exclusively to the monomeric form [15]. Dimerization of Eps8 might therefore be regarded as an “OFF” signal, which can be switched “ON” as the molecule becomes monomeric thereby allowing protein–protein interactions to occur. It should be noted, however, that the formation of a strand-exchanged dimeric Eps8–SH3 domain results in an extensive dimerization interface, much greater than usually observed for reversible regulated protein–protein associations in signal transduction. Thus, the physiological relevance of a dimer–monomer equilibrium in vivo remains to be established. The identification of the optimal binding peptides for the SH3 domain of Eps8 provided another unexpected result. In spite of the overall conservation of the primary structure and the similarity to the canonical SH3 fold, the SH3 domain of Eps8 binds preferentially to peptides containing a PXXDY, instead of the XPXXP, consensus sequence [15]. This binding specificity is conserved among the three Eps8-related genes that have been identified by screening an EST data bank [15]. Thus, the SH3 of Eps8 represents the prototype of a novel family of SH3 modules, which do not bind to canonical XPXXP containing peptides, and contract specific and distinct interactions in the signaling network. This contention is further supported by the isolation of two physiological interactors of the SH3 domain of Eps8, Abi1 and RN-tre, which display the PXXDY motif and, as it will be discussed later on, establish a novel network involved in the regulation of RTK-dependent signaling [1,12]. 3.3. Eps8 functional regions: the EGFR binding surface and the C-terminal “effector” region Structural–functional studies of Eps8 revealed the existence of two additional functional regions. The

first, encompassing amino acids 298–362, represents a binding surface for the juxtamembrane region of EGFR [2]. Albeit the functional consequences of this association have not been clarified, it is intriguing to speculate that it may contribute to recruitment of Eps8 and of Eps8-based complex to EGFR, thereby facilitating signal propagation. The second region, extending amino acids from 648 to 821, has been defined as a C-terminal “effector region” since it mediates a weak, but activating interaction with Sos1, a dual guanine nucleotide exchange factor for Ras and Rac protein, and a direct association to filamentous actin. Thus, it might participate both in facilitating the catalytic activation of the signaling complex, Eps8–Abi1–Sos1, and in directing Eps8 and Eps8-based complexes to their proper site(s) within the cell (see later and [18]). Notably, the Eps8 “effector region” overlaps with the SAM-PNT domain. However, the contribution of this putative domain to the function exerted by the C-terminal “effector region” of Eps8 remains to be established.

4. Eps8 coordinates and integrates multiple signaling pathways Signaling by and trafficking of RTKs involves small GTPases of the Rho and Rab families, respectively. Recently, a complex interplay between these two apparently distinct pathways has been revealed, and the molecular connectors of this circuitry are being identified. Eps8 has emerged as one of these molecular links capable of coordinating signals among GTPases involved in the control of actin dynamics and of receptor-mediated internalization. 4.1. Eps8 in RTK-activated signaling Among the Rho GTPases, Rac is essential in RTK-dependent modulation of actin [5]. Epistatic and biochemical analysis placed Rac downstream of Ras and PI3K, an enzyme that catalyzes the conversion of phosphatidylinositol 4,5 biphosphate to phosphatidylinositol 3,4,5 triphosphate (PIP3). Active RTKs bind to the p85 regulatory subunit of PI3K, thereby recruiting the enzyme to the plasma membrane, where it can phosphorylate its substrates [20]. In addition, Ras, in its active GTP-loaded state, may

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directly bind the catalytic subunit of PI3K, p110, thus concurring to its full activation. The product of PI3K activity, PIP3, is required to activate a number of Rac-specific Guanine Exchange Factors (GEF), such as Vav, Tiam-1 and Sos1, leading to activation of Rac [20]. Additional complexity is provided by the observation that Rac–GEF may assemble into macromolecular complexes, essential for efficient catalysis, proper subcellular localization and for signal coordination. One of these complexes comprises Eps8, Abi1, and Sos1 [17]. Within this complex, Abi1 serves as a scaffold bridging together Eps8 and Sos1. The ternary complex, inmmunoprecipitated from cells, displays Rac-specific GEF activity, which requires the presence of all three components. In addition, disruption of the complex by genetic removal of Eps8, abrogates Rac activation and Rac-dependent actin remodeling induced by RTKs, Ras or PI3K [17]. Sos1 acts as a bifunctional GEF since, it activates Ras when engaged in a complex with the adaptor molecules Grb2 [16], and as Rac–GEF, when present in a trimeric complex with Eps8 and Abi1 [6,17]. While the Sos1–Grb2 and Sos1–Abi1–Eps8 complexes coexist in the cells, they are differen-

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tially regulated by growth factor stimulation. The Eps8–Abi1–Sos1 complex is stable upon RTK activation while the Grb2–Sos1 complex is disrupted. This correlates with and may contribute to the different kinetics of activation of Ras, which is short lived, and Rac, which is sustained [6]. Thus, the engagement of Sos1 in two distinct, and differentially regulated molecular complexes dictates its catalytic specificity and may allow for co-ordinated activation of Ras and Rac and different duration of their signaling within the cell [6]. An additional level of complexity is highlighted by the finding that the C-terminal effector region of Eps8 binds to F-actin and directs Eps8 to sites where actin polymerization occurs, such as membrane ruffles [18], where Abi1 and Sos1 also localize [6]. Thus, the formation of the trimeric complex is required not only for its catalytic activation, but also to restrict and localize its activity in a defined microenvironment (Fig. 2). 4.2. Eps8 in endocytosis The GTPase Rab5 plays a critical role in the process of receptor internalization, possibly acting in the

Fig. 2. Eps8 integrates signals leading to actin cytoskeleton, via Rac, and receptor endocytosis, via Rab5. The involvement of Eps8 in multiple pathways is depicted, as obtained from a combination of biochemical, biological and genetic evidence. Black arrows indicate stimulatory effects. Red bars indicate inhibitory effects. The green line (with double arrowheads) indicates the existence of complexes in equilibrium. GTPase are in yellow, enzymes are in green, adaptor molecules are in orange, RTKs are in black and growth factors (G.F.) in light blue.

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initial phase of receptor sequestration into clathrincoated pits [22]. Furthermore, recent evidence indicates that RTK activation may regulate the activity of Rab5. First, EGF stimulation induces a specific and rapid activation of Rab5 by increasing the level of GTP-loaded Rab5 (reviewed in [22]). Second, signal emanating from activated RTK may also modulate Rab5 by regulating specific GAPs. This latter effect is likely mediated by RN-tre, a specific Rab5 GAP whose activity is downmodulated by EGF stimulation [10]. RN-tre, in turn displays features of an integrator of endocytosis and actin cytoskeleton. Its ectopic expression leads to inhibition of EGF and transferrin receptors endocytosis [10]. The former effect is, however, entirely dependent on the presence of Eps8, which binds RN-tre through its SH3 domain, suggesting a role of Eps8 in directing RN-tre to EGFR in the process of internalization. At the same time, binding of RN-tre to Eps8 affects the ability of the latter to bind to Abi1 (Abi1 and RN-tre compete for binding to the SH3 domain of Eps8), thereby diverting Eps8 from its actin dynamic regulatory function [10].

might both concur to the enhancement of receptor signaling.

4.3. Signal integration by Eps8

Acknowledgements

Available knowledge indicates that Eps8 integrates different signaling pathways by participating in actin remodeling through Rac, when in complex with Abi1 and Sos1, or in receptor endocytosis modulating Rab5 activity, when in complex with RN-tre (Fig. 2). A scenario can thus be envisioned whereby following activation of RTK, the Grb2–Sos1 complex is recruited to the plasma membrane permitting the exchange of guanine nucleotide on Ras. GTP-loaded Ras can bind to and contribute to the activation of its downstream effectors, such as PI3K. PI3K, with a mechanism still to be elucidated, signals to the Eps8–Abi1–Sos1 complex, and activates its Rac–GEF activity, thus leading to Rac activation and actin remodeling. Simultaneously, ligand-binding to RTK induces internalization of the receptors, a process that is also controlled by Rab5 activity. Modulation of Rab5 is, at least in part exerted through recruitment of the complex RN-tre–Eps8, which directly acts by catalyzing the hydrolysis of guanine nucleotide on activated Rab5, thereby inhibiting receptor internalization and prolonging receptor signaling at the plasma membrane. Thus, the Eps8–Abi1–Sos1 and Eps8–RN-tre complexes

Work in the authors’ labs is supported by grants from Associazione Italiana Ricerca sul Cancro to G.S and P.P.D.F, and from the Telethon Foundation, the CNR (Progetto Biotecnologie), and the EC (V framework) to P.P.D.F.

5. Perspectives The discovery of extensive cross talks among small GTPases highlights the importance of defining the molecular mechanisms through which different pathways activated by RTKs are integrated and co-regulated. Overwhelming evidence indicates that the subversion of this delicate and highly controlled signaling system is responsible for several pathologies, first and foremost cancer. Therefore, the knowledge of how signal integration is achieved is likely to provide the rationale to design novel strategies aimed at interfering with subverted signaling pathways. In this scenario, the exquisite specificity of the Eps8-based network (due to the unique binding properties of its SH3 domain) and its central role as an integrator of signals render Eps8 an appealing therapeutic target.

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