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• W2083504420 abstract "Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with theirDrosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis. Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with theirDrosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis. receptor tyrosine kinases mitogen-activated protein kinase epidermal growth factor epidermal growth factor receptor growth factor receptor-binding protein-2 Son of Sevenless fibroblast growth factor receptorsubstrate-2 Src homology domain-2 really interesting new gene Drosophila Sprouty murine Sprouty human Sprouty mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Drosophila Cbl glutathione S-transferase Ring finger phosphate-buffered saline hemagglutinin polyacrylamide gel electrophoresis ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein ligase extracellular signal-regulated kinase Receptor tyrosine kinases (RTKs)1 have been implicated in numerous cellular processes such as cell fate specification and differentiation (1Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), oncogenic transformation (2Porter A.C. Vaillancourt R.R. Oncogene. 1998; 17: R1343-R1352Crossref PubMed Scopus (277) Google Scholar), and axonal guidance (3Tan P.B. Kim S.K. Trends Genet. 1999; 15: 145-149Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Tyrosine residues in the cytoplasmic domains, which become rapidly phosphorylated following ligand engagement, orchestrate the intrinsic properties of RTKs. The activated RTK (or in some cases, an associated phosphotyrosine-bearing docker protein) thus serves as a docking site that attracts various signaling molecules to the membrane vicinity. The assembly of such signaling complexes allows the RTKs to initiate the transmission of signals from the membrane to the nucleus via the MAPK cascade (4Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (897) Google Scholar). This pathway uses a set of highly conserved signal transduction molecules to link the activated receptors to the MAPK cascade activator, the GTP-binding protein Ras (5Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: R1395-R1413Crossref PubMed Scopus (918) Google Scholar). The increasing number and complexity of proteins discovered to be involved in modulating the MAPK cascade indicate that the transmission of signals originating from the RTKs is under exquisite homeostatic control (6Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar).In the last decade, a number of major RTK/MAPK regulators have been isolated from genetic screens in developmental models, allowing for the delineation of many key mammalian signaling pathways. Fibroblast growth factor receptors and epidermal growth factor receptors (EGFRs) are subsets of RTKs that are coupled to Ras via one or more adaptor proteins that contain specific protein-protein interaction domains (7Plotnikov A.N. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar,8Moghal N. Sternberg P.W. Curr. Opin. Cell Biol. 1999; 11: 190-196Crossref PubMed Scopus (287) Google Scholar). In the EGFR signaling system, one such protein that serves as a direct link between the receptor and Ras is Grb2 (9Stern M.J. Marengere L.E. Daly R.J. Lowenstein E.J. Kokel M. Batzer A. Olivier P. Pawson T. Schlessinger J. Mol. Biol. Cell. 1993; 4: 1175-1188Crossref PubMed Scopus (53) Google Scholar), which binds constitutively to and recruits the Ras-activating guanine nucleotide exchange factor Sos (10Li N. Batzer A. Daly R. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar). Ras is subsequently activated by Sos via GDP/GTP exchange. Ras has an inherent, weak GTPase activity. Auxiliary proteins with GTPase-activating properties interact with and enhance the GTPase activity of Ras and hence are down-regulators of MAPK signaling pathways (11Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar). Fibroblast growth factor receptors activate the MAPK cascade by essentially a similar mechanism as EGFR, except they make use of a surrogate receptor cytosolic domain in the form of the constitutively associated FRS2 docker protein (12Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, 13Rabin S.J. Cleghon V. Kaplan D.R. Mol. Cell. Biol. 1993; 13: 2203-2213Crossref PubMed Scopus (173) Google Scholar, 14Lim Y.P. Low B.C. Ong S.H. Guy G.R. J. Biol. Chem. 1997; 272: 29892-29898Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), where tyrosine residues on FRS2 serve as substrates of fibroblast growth factor receptor and attract the SH2 domains of Grb2 and Shp2.A common component of many signaling modules is the multi-adaptor protein Cbl, first identified as a retroviral transforming gene product that induces pre-B cell lymphoma and myeloid leukemia (15Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (281) Google Scholar). The product of the mammalian c-cbl gene (p120Cbl) is a widely expressed cytoplasmic protein with several distinctive domains, including an SH2 domain, a Ring finger motif, and a large proline-rich stretch at its C terminus (16Meng W. Sawasdikosol S. Burakoff S.J. Eck M.J. Nature. 1999; 398: 84-90Crossref PubMed Scopus (246) Google Scholar). Genetic and biochemical studies have implicated c-Cbl in the attenuation of RTK-mediated signaling cascades; partial loss-of-function mutations of Caenorhabditis elegansLET-23 (an EGFR equivalent) result in developmental defects that are reversed by mutation of the c-Cbl ortholog SLI-1, which acts early at the level of LET-23, and the Grb2 homolog Sem5 (15Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (281) Google Scholar, 17Jongeward G.D. Clandinin T.R. Sternberg P.W. Genetics. 1995; 139: 1553-1566Crossref PubMed Google Scholar). The engagement of a variety of transmembrane receptors, including growth factor, antigen, and integrin receptors, results in tyrosine phosphorylation of c-Cbl and its association with numerous cytoplasmic signaling proteins (18Thien C.B. Langdon W.Y. Oncogene. 1997; 15: 2909-2919Crossref PubMed Scopus (57) Google Scholar, 19Liu Y.C. Altman A. Cell. Signal. 1998; 10: 377-385Crossref PubMed Scopus (84) Google Scholar). Recently, c-Cbl has been demonstrated to elevate the rate of ligand-induced endocytosis (termed “down-regulation”) of EGFR by tagging the receptors with ubiquitin and targeting them for destruction by the lysosomal/proteasomal system (20Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar).In Drosophila, Sprouty (dSPRY) was first identified as a down-regulator of the “Breathless” (the Drosophilaequivalent of fibroblast growth factor receptor) signaling cascade that governs proper tracheal branching (21Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). The removal or loss-of-function mutations of dSPRY gave rise to a morphological “sprouting” effect. dSPRY was also isolated in a separate genetic screen for inhibiting DER (the Drosophila equivalent of EGFR)-dependent cell recruitment during eye development (22Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar) and has been reported to be expressed in the developing eye imaginal disc and other tissues where EGFR signaling is known to exert its control (23Kramer S. Okabe M. Hacohen N. Krasnow M.A. Hiromi Y. Development. 1999; 126: 2515-2525Crossref PubMed Google Scholar). The C-terminal half of dSPRY was shown to localize the protein to the inner surface of the plasma membrane of Drosophila S2 cells, whereas the N-terminal portion interacted, at least in vitro, with DRK (the Drosophila homolog of Grb2) and Gap1, a Ras GTPase-activating protein (22Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Sequestration of a docking protein like DRK would constitute a plausible mechanism for inhibition of the early phase of RTK signaling. Gap1 has been shown previously to be a negative regulator of signaling for the Drosophila Sevenless RTK (24Gaul U. Mardon G. Rubin G.M. Cell. 1992; 68: 1007-1019Abstract Full Text PDF PubMed Scopus (235) Google Scholar). It is possible that dSPRY might interact with and augment the GTPase activity of Gap1, thus acting as an attenuator of Ras activity. More recent genetic evidence led to the postulation that dSPRY intercepts the Ras/MAPK cascade downstream of Ras, at the level of Raf or MEK (25Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar). Composite studies thus suggest that dSPRY acts as a general antagonist of the RTK signaling pathways (23Kramer S. Okabe M. Hacohen N. Krasnow M.A. Hiromi Y. Development. 1999; 126: 2515-2525Crossref PubMed Google Scholar, 25Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar).To date, four mammalian Sprouty homologs have been cloned, with no known binding motifs or physiological functions (21Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 26de Maximy A.A. Nakatake Y. Moncada S. Itoh N. Thiery J.P. Bellusci S. Mech. Dev. 1999; 81: 213-216Crossref PubMed Scopus (157) Google Scholar). The Sprouty proteins are classified under the same gene family by virtue of their characteristic cysteine-rich residues located in their carboxyl termini. Parallel investigations on the role of murine SPRY2 (mSPRY2) in the development of the embryonic mouse lung suggest a conservation of function between dSPRY and mSPRY2 with respect to their negative modulation of respiratory organogenesis (27Tefft J.D. Lee M. Smith S. Leinwand M. Zhao J.S. Bringas Jr., P. Crowe D.L. Warburton D. Curr. Biol. 1999; 9: 219-222Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Furthermore, overexpression of Sprouty constitutes a reduction in fibroblast growth factor-induced limb bud outgrowth (28Minowada G. Jarvis L.A. Chi C.L. Neubuser A. Sun X. Hacohen N. Krasnow M.A. Martin G.R. Development. 1999; 126: 4465-4475Crossref PubMed Google Scholar). We have also recently identified a novel translocation domain that is responsible for the general targeting of Sprouty proteins to membrane ruffles upon fibroblast growth factor/EGF stimulation (29Lim J.M. Wong E.S.M. Ong S.H. Yusoff P. Low B.C. Guy G.R. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We were interested in further characterizing hSPRY2 and its possible involvement in RTK signal down-regulation by identifying cellular proteins that interact with hSPRY2. We demonstrate in this study that hSPRY2 and dSPRY interact directly with c-Cbl and dCbl and that the hSPRY2-c-Cbl association leads to an inhibition of the role of c-Cbl in enhancing the rate of internalization and possibly the down-regulation of EGFR.DISCUSSIONRecently, the Cbl family proteins have attracted a considerable amount of attention as down-regulators of both RTK and non-RTK signaling (34Ota Y. Samelson L.E. Science. 1997; 276: 418-420Crossref PubMed Scopus (225) Google Scholar, 39Smit L. Borst J. Crit. Rev. Oncog. 1997; 8: 359-379Crossref PubMed Google Scholar). Genetic analysis implicated the orthologs of Cbl inC. elegans and Drosophila in down-regulating the EGFR growth-promoting function (17Jongeward G.D. Clandinin T.R. Sternberg P.W. Genetics. 1995; 139: 1553-1566Crossref PubMed Google Scholar, 20Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar, 38Meisner H. Daga A. Buxton J. Fernandez B. Chawla A. Banerjee U. Czech M.P. Mol. Cell. Biol. 1997; 17: 2217-2225Crossref PubMed Scopus (118) Google Scholar). Human c-Cbl has been reported in a number of cell systems to be tyrosine-phosphorylated upon receptor stimulation and was found to exert a negative regulatory role in tyrosine kinase signaling, albeit by an as yet undefined mechanism (31Galisteo M.L. Dikic I. Batzer A.G. Langdon W.Y. Schlessinger J. J. Biol. Chem. 1995; 270: 20242-20245Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 32Fukazawa T. Miyake S. Band V. Band H. J. Biol. Chem. 1996; 271: 14454-14459Abstract Full Text Full Text PDF Scopus (144) Google Scholar, 33Tanaka S. Neff L. Baron R. Levy J.B. J. Biol. Chem. 1995; 270: 14347-14351Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The N-terminal fragment of c-Cbl essentially consists of two functional domains: an unconventional SH2-like domain (which incorporates the four-helix bundle and EF-hand) and a Ring finger motif. Structure-based mutation studies in the four-helix bundle, EF, and SH2 domains revealed that the three domains together form an integrated phosphoprotein recognition module (16Meng W. Sawasdikosol S. Burakoff S.J. Eck M.J. Nature. 1999; 398: 84-90Crossref PubMed Scopus (246) Google Scholar). Furthermore, a critical role of the SH2 domain in c-Cbl function is demonstrated by the localization of a loss-of-function mutation in SLI-1 (C. elegans homolog of Cbl) within a 17-amino acid deletion N-terminal to the Ring finger (18Thien C.B. Langdon W.Y. Oncogene. 1997; 15: 2909-2919Crossref PubMed Scopus (57) Google Scholar); this structural alteration renders the 70Z-Cbl mutant oncogenic and causes it to exhibit an enhanced level of tyrosine phosphorylation as well as to abrogate the negative regulatory function of wild-type c-Cbl. In our binding domain analysis, we showed that neither disruption nor lack of the SH2 domain (Cbl-NSand Cbl-NE, respectively) or deletion of the 70Z region in the c-Cbl C terminus (Cbl-CR) was accountable for any lack of association with hSPRY2. We have delineated the hSPRY2-binding region specifically to the Ring finger domain of c-Cbl.Various groups have reported evidence that c-Cbl down-regulates growth factor receptors by helping to ubiquitinate them, thereby marking them for destruction by the lysosomal/proteasomal route (40Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 41Lee P.S.W. Wang Y. Dominguez M.G. Yeung Y.-G. Murphy M.A. Bowtell D.D.L. Stanley E.R. EMBO J. 1999; 18: 3616-3628Crossref PubMed Scopus (251) Google Scholar, 42Miyake S. Lupher M.L. Druker B. Band H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7927-7932Crossref PubMed Scopus (236) Google Scholar). More recently, the Ring finger domain of Cbl, an evolutionarily conserved structure present in >200 proteins, was found to possess E3 ubiquitin ligase activity (35Barinaga M. Science. 1999; 286: 223-224Crossref PubMed Scopus (5) Google Scholar, 43Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (909) Google Scholar). Conjugation of ubiquitin proceeds via a three-step mechanism (44Hershko A. Ciechanover A.L. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6792) Google Scholar). Initially, the ubiquitin-activating enzyme (E1) activates the C-terminal glycine residue of ubiquitin to a high energy thiol ester with an internal E1 cysteine residue. One of several ubiquitin-conjugating enzymes (E2) transfers the activated ubiquitin to the substrate that is specifically bound to a member of the E3 ubiquitin ligase family. E3 ligases catalyze the covalent attachment of ubiquitin to the substrate. In one case, tyrosine residues on activated EGFR become phosphorylated upon growth factor stimulation and bind to the SH2 domain of c-Cbl; c-Cbl functions as an E3 to mediate endocytic sorting of the target substrate by relaying activated ubiquitin molecules to EGFR via its Ring finger. Although all three members of the Cbl family (c-Cbl, Cbl-b, and Cbl-3) can enhance ubiquitination (40Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar), two oncogenic variants (70Z-Cbl and v-Cbl) whose Ring fingers are defective are unable to desensitize EGFR (18Thien C.B. Langdon W.Y. Oncogene. 1997; 15: 2909-2919Crossref PubMed Scopus (57) Google Scholar, 20Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar). The oncogenic viral counterpart (v-Cbl), which lacks a functional Ring finger, inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. This has exciting implications because, in relation to our findings, binding of hSPRY2 to the catalytic site of c-Cbl would suggest an important modulatory role of hSPRY2 in the fate and signaling potency of growth factor receptors. Additionally, we found that hSPRY2 binding to the Ring finger domain of c-Cbl abrogates the latter's ability to induce down-regulation of EGFR.Our studies have demonstrated that hSPRY2 binds directly to the Ring finger domain of c-Cbl (and possibly also Cbl-b) via a small N-terminal region. A similar observation was made for dSPRY and dCbl, the binding of which encompasses similar regions. This is indicative of a conservation of the interaction domains throughout evolution. In this respect, it is interesting to note that there is no Sprouty ortholog found in C. elegans. Taken together, the constitutive nature of the hSPRY2-c-Cbl association may highlight the requirement for some extrinsic factors to displace hSPRY2 from the Ring finger domain of c-Cbl and, by so doing, uplifts its suppressive effect on the latter.A number of questions are posed by the data presented. First of all, do the other mammalian Sprouty proteins also bind to the Ring finger of c-Cbl? Preliminary analysis revealed that murine SPRY1 binds c-Cbl, but not murine SPRY4 (data not shown). Hence, one might infer that mSPRY4 plays a different functional role from mSPRY1 and mSPRY2. We have recently presented evidence that the conserved cysteine-rich C-terminal domain in various Sprouty proteins is responsible for directing the proteins to membrane ruffles when cells are stimulated with growth factors (29Lim J.M. Wong E.S.M. Ong S.H. Yusoff P. Low B.C. Guy G.R. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Given the high degree of conservation among the C-terminal regions, this would suggest that Sprouty proteins have a common target for membrane ruffle association. We further demonstrated that c-Cbl colocalizes with hSPRY2 in coexpressing cells, but not with the non-binding hSPRY2ΔN11 mutant. It becomes interesting then to question the roles of hSPRY2 and c-Cbl in each of the two localities. Does hSPRY2 translocate to ruffles to assume a different function by virtue of different or additional binding partners, or does the hSPRY2-mediated translocation serve to target c-Cbl in near proximity to the membrane-anchored receptors? If the latter case is true, one would expect hSPRY2 to synergistically promote the action of c-Cbl in the ligand-induced down-regulation of EGFR, which differs from our experimental observation. Although we do not know the proportion of endogenous c-Cbl that is bound by hSPRY2 (and therefore “nonfunctional” in terms of EGFR down-regulation), our results suggest that the inhibition of EGFR internalization that we observed in c-Cbl- and hSPRY2-coexpressing cells may be due to an exclusion of prospective E2·ubiquitin complexes from the Ring finger domain of c-Cbl, thereby preventing downstream ubiquitination events. Alternatively, hSPRY2 may have a direct involvement in receptor endocytosis.On the other hand, we cannot rule out the possibility that a fraction of the c-Cbl population that hSPRY2 binds to is tyrosine-phosphorylated or not or that, upon growth factor stimulation, hSPRY2 initiates a stronger preference for binding to other protein partners that might mediate translocation of the hSPRY2·c-Cbl complex to ruffles. It is interesting to note from the immunofluorescence studies on hSPRY2- and c-Cbl-coexpressing cells that a significant pool of c-Cbl still retains an association with microtubules in the cytosol, although a major portion has been shown to colocalize with hSPRY2 at the membrane ruffles in EGF-treated cells. Given the previously reported function of dSPRY and mSPRY2 in the attenuation of RTK activation (21Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 22Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 27Tefft J.D. Lee M. Smith S. Leinwand M. Zhao J.S. Bringas Jr., P. Crowe D.L. Warburton D. Curr. Biol. 1999; 9: 219-222Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), it is also possible that hSPRY2 associates with other signaling proteins to down-regulate the MAPK cascade. It will be interesting to pursue any substantial attenuation of MAPK signaling in hSPRY2- and c-Cbl-coexpressing cells. It is noteworthy that other studies done by overexpressing two forms of c-Cbl mutants (namely C381A and 70Z-Cbl) showed an inhibitory effect on EGFR down-regulation, but no effect on ERK activity was detectable (18Thien C.B. Langdon W.Y. Oncogene. 1997; 15: 2909-2919Crossref PubMed Scopus (57) Google Scholar, 37Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar).Do the various Sprouty isoforms bind to the relatively well conserved Ring finger domains of proteins other than c-Cbl? Despite the increasing number of proteins known to be ubiquitinated, the identification of the corresponding ubiquitin ligases (E3) has been lagging. E3 ligases for which the amino acid sequences are known include the N-end rule E3 ligases of yeast and mammals (45Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar); members of the HECT (homologous to E6-APCterminus) family (46Schwarz S.E. Rosa J.L. Scheffner M. J. Biol. Chem. 1998; 273: 12148-12154Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar); Mdm2 (47Honda R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (308) Google Scholar); APC, theanaphase-promoting complex (48Page A.M. Hieter P. Annu. Rev. Biochem. 1999; 68: 583-609Crossref PubMed Scopus (131) Google Scholar); and other F-box- and cullin-containing complexes (49Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar). In the last few years, the once discrete Ring finger motifs intrinsic to a large number of proteins have surfaced as instrumental components that confer on E3 proteins a capacity for E2-dependent ubiquitination (50Saurin A.J. Borden K.L.B. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-215Abstract Full Text PDF PubMed Scopus (609) Google Scholar,51Lorick K.L. Jensen J.P. Fang S.Y. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Scholar). In view of this, the mode of action of Sprouty as a general RTK inhibitor certainly encompasses several layers of complexity.With the above possibilities and taking current evidence into account, it is difficult to envisage a simplistic model to explain how the interaction between Sprouty and Cbl manifests itself in the down-regulation of RTK signaling. In addition to the observed binding between hSPRY2 and Cbl-b, which has been shown to play other distinct roles in EGF-mediated signaling (52Ettenberg S.A. Keane M.M. Nau M.M. Frankel M. Wang L.M. Pierce J.H. Lipkowitz S. Oncogene. 1999; 18: 1855-1866Crossref PubMed Scopus (102) Google Scholar), there are likely to be other candidate proteins that interact with Sprouty and Cbl. We are screening and characterizing those proteins that bind to the various Sprouty family members in an effort to better understand their physiological function. Receptor tyrosine kinases (RTKs)1 have been implicated in numerous cellular processes such as cell fate specification and differentiation (1Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), oncogenic transformation (2Porter A.C. Vaillancourt R.R. Oncogene. 1998; 17: R1343-R1352Crossref PubMed Scopus (277) Google Scholar), and axonal guidance (3Tan P.B. Kim S.K. Trends Genet. 1999; 15: 145-149Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Tyrosine residues in the cytoplasmic domains, which become rapidly phosphorylated following ligand engagement, orchestrate the intrinsic properties of RTKs. The activated RTK (or in some cases, an associated phosphotyrosine-bearing docker protein) thus serves as a docking site that attracts various signaling molecules to the membrane vicinity. The assembly of such signaling complexes allows the RTKs to initiate the transmission of signals from the membrane to the nucleus via the MAPK cascade (4Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (897) Google Scholar). This pathway uses a set of highly conserved signal transduction molecules to link the activated receptors to the MAPK cascade activator, the GTP-binding protein Ras (5Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: R1395-R1413Crossref PubMed Scopus (918) Google Scholar). The increasing number and complexity of proteins discovered to be involved in modulating the MAPK cascade indicate that the transmission of signals originating from the RTKs is under exquisite homeostatic control (6Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar). In the last decade, a number of major RTK/MAPK regulators have been isolated from genetic screens in developmental models, allowing for the delineation of many key mammalian signaling pathways. Fibroblast growth factor receptors and epidermal growth factor receptors (EGFRs) are subsets of RTKs that are coupled to Ras via one or more adaptor proteins that contain specific protein-protein interaction domains (7Plotnikov A.N. Schlessinger J. Hubbard S.R. Mohammadi M. Cell. 1999; 98: 641-650Abstract Full Text Full Text PDF PubMed Scopus (502) Google Schol" @default.
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