Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling
2002; Springer Nature; Volume: 21; Issue: 18 Linguagem: Inglês
10.1093/emboj/cdf493
ISSN1460-2075
AutoresEsther Sook Miin Wong, Chee Wai Fong, Jormay Lim, Permeen Yusoff, Boon Chuan Low, Wallace Y. Langdon, Graeme R. Guy,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoArticle16 September 2002free access Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling Esther Sook Miin Wong Esther Sook Miin Wong Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Chee Wai Fong Chee Wai Fong Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Jormay Lim Jormay Lim Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Permeen Yusoff Permeen Yusoff Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Boon Chuan Low Boon Chuan Low Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543 Search for more papers by this author Wallace Y. Langdon Wallace Y. Langdon Department of Pathology, M Block, Room 1.2, Queen Elizabeth II Medical Centre, University of Western Australia, WA, Australia Search for more papers by this author Graeme R. Guy Corresponding Author Graeme R. Guy Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Esther Sook Miin Wong Esther Sook Miin Wong Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Chee Wai Fong Chee Wai Fong Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Jormay Lim Jormay Lim Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Permeen Yusoff Permeen Yusoff Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Boon Chuan Low Boon Chuan Low Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543 Search for more papers by this author Wallace Y. Langdon Wallace Y. Langdon Department of Pathology, M Block, Room 1.2, Queen Elizabeth II Medical Centre, University of Western Australia, WA, Australia Search for more papers by this author Graeme R. Guy Corresponding Author Graeme R. Guy Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 Search for more papers by this author Author Information Esther Sook Miin Wong1, Chee Wai Fong1, Jormay Lim1, Permeen Yusoff1, Boon Chuan Low2, Wallace Y. Langdon3 and Graeme R. Guy 1 1Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore, 117609 2Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543 3Department of Pathology, M Block, Room 1.2, Queen Elizabeth II Medical Centre, University of Western Australia, WA, Australia *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4796-4808https://doi.org/10.1093/emboj/cdf493 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF- mediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2ΔN11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation. Introduction Growth factor-induced signalling by receptor tyrosine kinases (RTKs) plays a central role in: embryonic development, signalling in mature cells and regulation of pathogenesis. Various regulatory proteins and feedback mechanisms tightly control these key pathways. Drosophila Sprouty (dSpry) was originally identified as an antagonist of Drosophila development-associated RTK signalling. In dSpry mutants, signalling via the fibroblast growth factor receptor (FGFR) in lung development is deregulated and excessive tracheal branching is observed (Hacohen et al., 1998). dSpry is also expressed in the developing eye and other tissues under the control of epidermal growth factor receptor (EGFR), where it acts to attenuate downstream signalling. Casci et al. (1999) identified dSpry to be an intracellular protein that interacts in vitro with Drk (Drosophila homologue of Grb2) and Gap-1, a Ras GTPase-activating protein, to potentially inhibit Ras signalling. Currently, four mammalian genes have been identified that encode protein homologues of dSpry (de Maximy et al., 1999; Minowada et al., 1999); mSpry2 is ubiquitously expressed in adult tissues such as the brain, kidney, lung, heart and skeletal muscle (Tefft et al., 1999). The various Spry isoforms have variable N-terminal sequences but share considerable cysteine-rich sequence homology in their C-termini. This shared domain appears to play a role in protein targeting to foci in the membrane periphery (ruffles) when cells are stimulated by epidermal growth factor (EGF) or fibroblast growth factor (FGF) (Lim et al., 2000). The relatively poorly conserved N-terminal halves of the proteins are assumed to interact with other effectors to manifest the putative inhibitory role of Spry. The conserved function of dSpry and mammalian Sprys in the negative regulation of growth factor-induced signalling has been documented (Kramer et al., 1999; Reich et al., 1999). Based on genetic evidence, several molecular mechanisms by which Spry proteins might inhibit RTK signalling have been postulated; dSpry acts to inhibit FGF signalling at a point between the activated receptor and Ras (Casci et al., 1999), or at the level of Raf (Reich et al., 1999). Additionally, evidence has been presented that indicates that mSpry4 regulates angiogenesis by inhibition of the FGF pathway upstream of Ras (Lee et al., 2001), and mSpry2 functions as a negative regulator of embryonic lung morphogenesis and development (Mailleux et al., 2001). The mitogen-activated protein kinase (MAPK) cascade is a central intracellular signalling pathway linking activation of surface receptors to cytoplasmic and nuclear effectors by transducing signals from RTKs to two members of the Ras family of small G-proteins, Ras and Rap1 (Marshall, 1998). Ras interacts directly with, and sequentially activates Raf serine/threonine kinases (c-Raf and B-Raf); phosphorylated Raf then activates MEK, which in turn phosphorylates and activates the MAPKs (ERK1 and ERK2). ERK1/2 have been shown to be essential for cellular proliferation, as well as phenotypic determination (Marshall, 1995). One of the best-studied model systems employed to examine MAPK responses in determining cellular phenotype is the proliferation/differentiation-responsive rat pheochromocytoma (PC12) cell line. These cells respond to EGF treatment by increasing proliferation. In contrast, FGF and nerve growth factor (NGF) stimulation of PC12 cells results in their differentiation into a neuron-like phenotype (Kao et al., 2001). There is well-supported evidence that the amplitude and longevity of MAPK signal governs whether these cells are stimulated to proliferate, or to withdraw from the cell cycle and differentiate. EGF and other mitogens are reported to invoke transient activation of the MAPKs, whereas FGF and NGF treatment results in sustained activation of this signalling pathway. The ErbB family of RTKs exemplifies the importance of stringent regulation on signalling potency and its impact on human cancers (Yarden and Sliwkowski, 2001). Of the four ErbB family members, ErbB-1 (or EGFR) has the unique property of binding to the negative regulator c-Cbl, and is effectively targeted for lysosomal and proteasomal degradation (Levkowitz et al., 1998). Current concepts regarding c-Cbl function indicate that it is a complex adaptor protein whose phosphorylation leads to formation of activation-dependent complexes (Thien and Langdon, 2001). Despite an incomplete knowledge of its mechanism of action, it is apparent that c-Cbl promotes polyubiquitylation and hence degradation (or ‘down-regulation’) of certain ligand-activated RTKs (e.g. PDGFRα; Miyake et al., 1998), of which extensive studies have been made with EGFR (Levkowitz et al., 1998). The ubiquitin system defines the recruitment of ubiquitin-conjugating enzymes (E2) to specific proteins recognized for degradation through an E3 ubiquitin ligase (Hershko and Ciechanover, 1998). c-Cbl functions as an E3 that docks on to phosphotyrosine sites on activated substrates (via its unique SH2 domain), bringing them into close proximity with an E2 enzyme (bound via its intrinsic RING finger domain) for the relay of activated ubiquitin moieties to target proteins, thereby marking them for later lysosomal/proteasomal destruction and consequent signal attenuation (Joazeiro et al., 1999). The effects of c-Cbl-mediated ubiquitylation on EGFR endocytosis are currently enigmatic. Some groups have demonstrated that c-Cbl-mediated EGFR ubiquitylation does not enhance the endocytic rate of receptors, but expression of inactive RING finger mutants (C381A and ΔRF) inhibits receptor ubiquitylation and subsequent endocytosis (Levkowitz et al., 1998; Waterman et al., 1999). Furthermore, despite possessing intact SH2 domains, two known oncogenic mutants of c-Cbl are both incapable of facilitating ubiquitylation due to their failure to recruit the ubiquitin system to activated RTKs, since they either lack all (v-Cbl) or part (70Z-Cbl) of the RING finger (Levkowitz et al., 1999). The objective of this study was to investigate the functional effect of hSpry2 on the regulation of MAPK by EGF in relation to the disposition of the ligand-bound receptors, as well as to address the implications of hSpry2-c-Cbl association in mediating this outcome. Results hSpry2 prevents internalization of EGFRs into endocytic vesicles Previously we have demonstrated that a sequence spanning amino acids 11–53 at the N-terminal end of hSpry2 interacts directly with the RING finger domain of c-Cbl (Wong et al., 2001). A preliminary experiment indicated that a corollary of this interaction was increased numbers of EGFRs being retained at the cell surface. Prior to assessing the likely downstream effects of such an event, we sought to apply alternative and more informative techniques to ascertain the extent and nature of EGFR surface retention. Confocal microscopy was employed to observe visually the relationship between EGFR, c-Cbl and isoforms of Spry. We performed a time-dependent (0, 2, 5, 10, 20 and 30 min EGF stimulation) observation on the subcellular localization of EGFRs, in the presence or absence of overexpressed hSpry2 (10 min time-points shown in Figure 1). In unstimulated COS-1 cells, both c-Cbl and EGFR display diffused, cytosolic disposition across the whole cell interior except for the nuclei, with peripheral lining of EGFR at the plasma membrane; the staining pattern of EGFR was unchanged in resting cells transfected with hSpry2 (data not shown). In EGF-stimulated control cells, c-Cbl staining shows a punctate pattern indicative of its incorporation into endocytic vesicles (Figure 1A and C), consistent with the observation that ligand stimulation induces a rapid subcellular relocalization of c-Cbl (Levkowitz et al., 1998). In those same cells, EGFRs can also be observed within endocytic vesicles, as identified by staining with antibodies against EEA1, an established vesicle marker protein (Mu et al., 1995; data not shown), with increased numbers on the cell periphery (Figure 1B and C). With transiently transfected hSpry2, which has been established to translocate to membrane ruffles upon EGF stimulation (Figure 1D), there appears to be a near total inhibition of EGFR endocytosis in that the receptors were mainly retained at the membrane surface and were not apparent in endocytic vesicles throughout the experimental time course of EGF stimulation (10 min time-point shown, Figure 1E and F). Individual N- and C-terminal domains of hSpry2 were found to be incapable of inhibiting receptor endocytosis (data not shown). Hence, the ability of hSpry2 to bind the RING finger of c-Cbl is clearly insufficient to function autonomously as an inhibitor of EGF-induced endocytosis. Seemingly, specific intracellular localization provided by the putative translocation domain in the C-terminal half of hSpry2 (Lim et al., 2000) is necessary for its optimal function. Thus, it would appear that a full-length protein that can bind c-Cbl as well as be targeted to specific membrane locations (ruffles) is essential for withholding the majority of EGFRs at the cell surface and preventing their subsequent transition into endocytic vesicles. We further examined the effects of hSpry2ΔN11 and mSpry4, neither of which binds c-Cbl, on the fate of EGFR localization. In both cases, receptor internalization was not blocked upon EGF stimulation and EGFRs were apparent in punctate endosomal structures, with traceable surface receptor staining (Figure 1G–J). Figure 1.hSpry2 specifically inhibits EGFR endocytosis. COS-1 cells were singly transfected with 1 μg each of HA-tagged c-Cbl alone or FLAG-tagged Sprys, and subjected to serum-deprivation overnight prior to stimulation with 100 ng/ml EGF at 37°C for 10 min. Cells were then fixed, permeabilized and stained for endogenous EGFR using an anti-EGFR monoclonal and FITC-conjugated AffiniPure rabbit anti-mouse IgG (green) secondary antibody. c-Cbl was visualized with an anti-c-Cbl polyclonal, and FLAG-tagged Spry constructs were detected using a polyclonal anti-FLAG, followed by Texas Red dye-conjugated AffiniPure goat anti-rabbit IgG (red). Merged signals are labelled as overlay staining (yellow). Bar = 10 μm. Download figure Download PowerPoint Increased cell surface retention of EGFRs with hSpry2 expression The process of receptor endocytosis has many phases and it was of interest to investigate at which stage EGFR endocytosis was inhibited by hSpry2 overexpression: on the cell surface or at different stages of incorporation into vesicles or internalization of nascent vesicles. We therefore employed an analytical method that measures only cell surface EGFRs following ligand-mediated endocytosis. Initial down-regulation of EGFRs was induced using an unlabelled ligand and status of the remaining surface-associated binding sites was then determined by performing a direct radiolabelled EGF–receptor binding assay. From the graphical representation in Figure 2A, the statistics of external EGFRs in unstimulated cells from various transfection combinations were comparable. However, the patterns of receptor down-regulation exhibited by EGF-treated transfectants were different: whereas c-Cbl accelerates the degree of EGFR down-regulation, the ubquitylation-defective mutant c-Cbl-C381A was unable to mediate such an effect and high levels of EGFRs were retained at the cell surface. A similar phenomenon was noted in hSpry2-transfected cells, but not with the c-Cbl non-binding (hSpry2ΔN11 or mSpry4) transfectants, where the surface EGFR levels were observed to decrease normally, although their effects were not as complete as that exhibited by wild-type c-Cbl. Figure 2.hSpry2 enhances cell surface retention of EGFRs. (A) To quantify the surface EGFR population, COS-1 cells were co-transfected with 0.1 μg of an EGFR expression vector, together with 0.5 μg plasmid encoding c-Cbl alone (pink triangles) or with either 0.4 μg FLAG–hSpry2 (red squares), FLAG–hSpry2ΔN11 (blue squares) or FLAG–mSpry4 (yellow squares) cDNA, or with 1.0 μg plasmid encoding the RING finger-defective form of c-Cbl (C381A) alone (green triangles). Forty-eight hours post-transfection, serum-starved culture cells were subjected to stimulation without or with 100 ng/ml EGF at 37°C for the time periods indicated. Bound EGF was removed, and the level of surface receptors relative to the initial number of ligand-binding sites was determined by incubating sister cultures with [125I]EGF (10 ng/ml) at 4°C for 2–4 h, in the absence or presence of a 100-fold excess of unlabelled EGF (1 μg/ml). Control cells were not exposed to EGF (circles). The results are expressed as the average fraction of original binding sites that remained on the cell surface after exposure to the unlabelled ligand at 37°C. The graphical plot shown is derived from a single experiment that is representative of four independent experiments. (B) Similar transfections (closed symbols) and treatment as in (A), except that cells were pre-incubated without or with 100 ng/ml EGF in the presence of 30 μM monensin at 4°C for 2 h before a temperature shift to 37°C for the various time periods. The residual level of surface-bound receptors that did not undergo down-regulation was then assayed by performing a direct binding assay with radiolabelled EGF. The average of duplicate determinations was expressed as the percentage of total radioactivity at time = 0 min. The experiment was repeated twice. Download figure Download PowerPoint Following an initial phase of receptor down-regulation, the reappearance of EGFRs was observed, presumably due to recycling of endocytosed receptors (see Figure 2A, 40–60 min time-points); that reappearance was completely inhibited by monensin (Figure 2B), a chemical ionophore known to inhibit recycling of transmembrane receptors (Basu et al., 1981) including EGFRs (Gladhaug and Chrisofferson, 1988). Despite monensin treatment, overexpression of hSpry2 effects a high retention of EGFRs at the cell surface (Figure 2B). These results indicate that hSpry2 inhibits EGFR endocytosis at a relatively early stage of the relocation/internalization process, but exerts no apparent effect on the fate of recycling receptors. 4β-phorbol 12-myristate 13-acetate (PMA) has been established previously to divert internalized EGFR molecules from a degradative fate to a recycling pathway (Bao et al., 2000). The dose of inhibitor administered was shown to be effective, where cells exposed to PMA in concert with monensin show significantly reduced levels of surface EGFRs compared with cells without monensin treatment (data not shown). EGFR ubiquitylation is abrogated in cells expressing hSpry2 Currently only a few receptor internalization signals have been described. Of these, ubiquitylation has been demonstrated to be a principal trigger for internalization of receptors in yeast cells (Hicke, 1997). Additionally, various effects have been ascribed to mono- and polyubiquitylation of receptors. The endocytosis of EGFRs has been comparatively well studied and c-Cbl, in its role as an E3 ubiquitin ligase, plays a critical role in this process. Since hSpry2 binds directly to the functional RING finger domain on c-Cbl that is necessary for the catalytic transfer of ubiquitin to substrate, it is plausible that hSpry2 affects the ubiquitylation status of EGFRs. We therefore examined whether regulation of the uptake of EGFRs into endosomes correlates with the degree of EGFR ubiquitylation. Ubiquitin cDNA was transfected into COS-1 cells in combination with either vector control, c-Cbl, c-Cbl-C381A mutant (which possesses a crucial disruption in the first cysteine residue of its RING finger and is thus incapable of ubiquitin ligase activity; Waterman et al., 1999) and various Sprys. Cell lysates were precipitated with anti-EGFR and analysed for the extent of ubiquitylation on EGFRs as well as equal expression of the transfected proteins (Figure 3A). Little or no ubiquitylation is detected in resting cells, and the immunoprecipitated EGFR amounts are comparable (data not shown). In the case of EGF-stimulated cells, EGFR ubiquitylation in vector-transfected control cells reflects an amount of ‘background ubiquitylation’ most likely due to endogenous c-Cbl. Overexpressed c-Cbl causes a substantial increase in the amount of ubiquitylated EGFRs, whereas overexpression of c-Cbl-C381A with a defective ubiquitin transferase catalytic site results in significantly less amounts. The levels of EGFR ubiquityl ation in cells co-transfected with mSpry4 or hSpry2ΔN11 are comparable to those in the vector-transfected control, as expected. Only hSpry2 is able to effectively abolish the observed ubiquitylation to a level similar to that induced by the inactive c-Cbl mutant, commensurate with its ability to bind the functional RING finger effector domain of c-Cbl. In the presence of c-Cbl there is enhanced internalization and degradation of EGFRs concomitant with increased ubiquitylation, as indicated by a drop in total immunoprecipitated EGFRs. This is in contrast to the effect obtained with hSpry2, in which much higher levels of residual EGFRs are observed that are not being ubiquitylated. Figure 3.hSpry2 abrogates c-Cbl-dependent ubiquitylation of EGFRs. (A) COS-1 cells were co-transfected with 1.0 μg HA-tagged ubiquitin and 3.0 μg each of either vector control, c-Cbl, c-Cbl-C381A or the different FLAG-tagged Spry forms per 100 mm culture dish. Forty-eight hours later, cell monolayers were treated with 100 ng/ml EGF at 37°C for 10 min. Total cell lysates (TCL) were subjected to immunoprecipitation (IP) using anti-EGFR–agarose-conjugated beads, immunoblotted (IB) with anti-HA to distinguish ubiquitin-conjugated EGFR, and anti-EGFR to assess the amounts of immunoprecipitated EGFR. TCL samples were analysed with anti-HA or anti-FLAG to show relative expression levels of the various constructs. The bracket indicates the position of high molecular weight species of ubiquitin-positive EGFRs. (B) Immunoprecipitated EGFR proteins were subjected to an in vitro ubiquitylation assay in the presence of purified UbcH7 (or no added UbcH7 as control without E2), eluted c-Cbl protein alone (or no added c-Cbl as control without E3), recombinant c-Cbl with either GST–hSpry2, GST–mSpry4 or GST–hSpry2ΔN11 fusion proteins, or c-Cbl-C381A fusion protein alone, plus the essential components in the ubiquitylation system. The reaction products were analysed following western blotting protocol with anti-ubiquitin. The bracket highlights the position of high molecular species of ubiquitin-positive EGFRs. (C) Competitive binding between hSpry2 and UbcH7 for the RING finger domain of c-Cbl. COS-1 cells (100 mm dishes) were co-transfected with 3.0 μg each of FLAG-tagged UbcH7, HA–c-Cbl or HA–c-Cbl-ΔRF mutant, and varying amounts of FLAG–hSpry2 or the c-Cbl non-binding truncation mutant FLAG–hSpry2ΔN11, as indicated. Serum-deprived cells were stimulated with 100 ng/ml EGF at 37°C for 10 min. TCL were subjected to precipitation using anti-HA and immunoblotted (IB) with anti-FLAG to detect associated hSpry2 or UbcH7, and anti-HA to show the relative amounts of immunoprecipitated c-Cbl. A TCL blot was probed with anti-FLAG to demonstrate quantitatively the expression levels of exogenous hSpry2 and UbcH7 proteins. (D) COS-1 cells (100 mm dishes) were untransfected (control), or co-transfected with 3.0 μg each of FLAG-tagged UbcH7 and either HA–hSpry2, HA–human Ariadne-2 (hARI-2), HA–Drosophila Ariadne-1 (dAri-1), HA–c-Cbl or HA–c-CblΔRF constructs. Immunocomplexes of FLAG–UbcH7 or ‘pull-downs’ using GST–hSpry2 were detected using anti-HA. Total cell lysates were analysed for equivalent levels of protein expression and normalized sample loading using antibodies as indicated. An immunoblot (with TCL loaded alongside) was probed with anti-HA to detect hSpry2-binding proteins, and with anti-FLAG to verify equal amounts of immunoprecipitated UbcH7. Download figure Download PowerPoint To substantiate our observation that hSpry2 prevents c-Cbl-mediated ubiquitylation of EGFRs, an in vitro ubiquitylation assay was performed to demonstrate a defined impediment to c-Cbl activity as an E3 ubiquitin ligase. In this experiment, a ubiquitylation reaction was reconstituted wherein EGFRs serve as substrates of c-Cbl in an in vitro enzyme mixture containing the essential ubiquitin system components: ubiquitin, E1, E2 (UbcH7), E3 (c-Cbl) and ATP, with addition of eluted glutathione S-transferase (GST)–hSpry2, GST–mSpry4 or GST– hSpry2ΔN11 proteins. As a negative control, recombinant protein of c-Cbl-C381A ubiquitylation-defective mutant was added in place of wild-type c-Cbl. From Figure 3B, heavy polyubiquitylation of substrate EGFRs was ob served with c-Cbl alone, and in the presence of mSpry4 and hSpry2N11; but not with hSpry2, which shows only a faint background smear comparable to the negative control (c-Cbl-C381A). hSpry2 competes with UbcH7 for specific binding to the c-Cbl RING finger Ubiquitylation is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or mono- ubiquitylation, respectively (Laney and Hochstrasser, 1999; Hicke, 2001). There is currently no direct evidence that c-Cbl is involved in monoubiquitylation. From the data presented, it is conceivable that hSpry2, by binding to the RING finger of c-Cbl (or another unidentified ubiquitin E3 ligase), is competing against the binding of ubiquitin E2 proteins and hence preventing subsequent polyubiquitylation of substrate proteins by c-Cbl. To investigate likely competition between a prospective E2 and hSpry2, COS-1 cells were transfected with a constant amount of UbcH7 (human E2) and HA–c-Cbl, together with an increasing dosage of hSpry2 or the c-Cbl non-binding hSpry2ΔN11 as indicated. HA–c-CblΔRF (with RING finger domain deleted) was included as a negative binding control for hSpry2 and UbcH7. Analysis of anti-HA immunoprecipitates revealed that the binding of UbcH7 to the c-Cbl RING finger is competed off by increasing amounts of hSpry2 (Figure 3C). It thus appears that when present in sufficient concentrations, hSpry2 has the capacity to inhibit receptor ubiquitylation by hindering the interaction between the E2 and E3 ubiquitin transferases. We also addressed whether the interaction between hSpry2 and the RING finger of c-Cbl is specific, as RING domains are well conserved in structure and have a commonality of function in the ubiquitylation process (Borden, 2000). Proteins other than c-Cbl, such as the Ariadne family of RING-containing proteins, recruit UbcH7 in a similar RING-dependent manner. HHAri, the human homologue of Drosophila Ariadne, has been reported to interact and co-localize with UbcH7 in mammalian cells via its RING finger domain (Ardley et al., 2001). Additionally, an interaction between the orthologues of HHAri and UbcH7 has been demonstrated in Drosophila (Aguilera et al., 2000). To investigate the specificity of hSpry2 binding and inhibitory effect on c-Cbl, we tested the abilities of dAri-1 and hAri-2 (a protein closely related to HHAri that can also interact with UbcH7) to substitute for c-Cbl binding to hSpry2. COS-1 cells were co-transfected with FLAG–UbcH7 and the various known binding or non-binding partners, as indicated in Figure 3D. Immunoprecipitated UbcH7 binds only to RING finger-containing proteins, indicating that the various constructs were functional in vivo. The binding specificity of hSpry2 to the RING finger-containing constructs was assessed in GST ‘pull-down’ assays (Figure 3D); hSpry2 interacts specifically with c-Cbl, but fails to bind either dAri-1 or hAri-2. Our findings are in accord with Ardley et al. (2001), who reported that the highly homologous UbcH7-interacting RING finger structures of HHAri and c-Cbl could not be functionally interchanged. The binding specificity of hSpry2 for the c-Cbl RING finger was further affirmed by its lack of in vitro and in vivo binding with Hakai, a recently identified c-Cbl-like E3 ubiquitin ligase (Fujita et al., 2002; data not shown). Decreased EGFR ubiquitylation upon expressing hSpry2 correlates with sustained ERK phosphorylation The well-characterized Ras/MAPK pathway is stimulated by a number of activated receptors including EGFRs (Gonzalez et al., 1991; Moghal and Sternberg, 1999). There has been considerable debate centred on whether receptor internalization occurs to propagate or terminate ERK signalling. In our experimental system we had
Referência(s)