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• W2070655784 abstract "Several genetic studies in Drosophilahave shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context. Several genetic studies in Drosophilahave shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context. Drosophila Sprouty fibroblast growth factor FGF receptor-1 FGFR substrate-2 epidermal growth factor EGF receptor receptor tyrosine kinase extracellular signal-regulated kinase mitogen activated protein MAP kinase/extracellular signal-regulated kinase kinase c-Jun N-terminal kinase growth factor receptor-binding protein-2 glutathione S-transferase hemagglutinin Drosophila Sprouty (dSpry)1 was discovered in genetic screens designed to detect novel genes involved in tracheal branching in the embryo (1Hacohen 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). Fibroblast growth factor (FGF) is one of the agents involved in this phase of embryonic development, and dSpry was deemed to be an inhibitor of FGF signaling pathways. Correlative evidence suggested that the protein may be secreted and act as a competitive inhibitor at the receptor level (1Hacohen 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). Another genetic screen, which analyzed genes associated with Drosophila eye development, further indicated that the inhibition by dSpry was not confined to FGF signaling but extended also to the epidermal factor receptor (EGFR) pathway. In contrast to the first study, dSpry was found to be an endogenous, membrane-located protein that inhibited the Ras/mitogen-activated protein (MAP) kinase pathway between receptor stimulation and the activation of Ras (2Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The authors also provided preliminary evidence that dSpry binds directly to Drk, the Drosophila equivalent of mammalian Grb2, and to the GAP-1 protein. Another genetic-based study also found dSpry to be an endogenously acting protein that inhibited the Ras/MAP kinase pathway downstream of Ras activation around the level of Raf (3Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar).While Drosophila has only one Spry protein, mammals have at least four isoforms (1Hacohen 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, 3Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar, 4de 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, 5Minowada 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). This family of proteins is characterized by a highly conserved cysteine-rich C-terminal half and variable N-terminal sequences. Evidence from mammalian systems indicates that mSpry2 has an inhibitory effect on lung alveoli branching that parallels the effects on the tracheal system of insects (6Tefft J.D. Lee M. Smith S. Leinwand M. Zhao J. Bringas Jr., P. Crowe D.L. Warburton D. Curr. Biol. 1999; 9: 219-222Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar).The MAP kinase cascades constitute highly conserved signaling systems that have been deemed to play various roles in physiological responses such as in cell growth, differentiation, oncogenic transformation, immune responses, and apoptosis (7Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1374) Google Scholar, 8Schaeffer H.J. Weber M. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1397) Google Scholar). The MAP kinase cascades are organized into core signaling modules consisting of three protein kinases: a MAP kinase kinase kinase (MKKK), a MAP kinase kinase (MKK), and a MAP kinase. Signals are transmitted through the module by sequential phosphorylation and activation of these component kinases (9Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1382) Google Scholar). The MAP kinase family is composed of several sub-families including the ERKs, c-Jun N-terminal kinase (JNK), and p38 MAP kinase (10Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 11Ferrell Jr., J.E. Trends Biochem. Sci. 1996; 21: 460-466Abstract Full Text PDF PubMed Scopus (486) Google Scholar). In the mammalian system, the ERK cascade relays signals from receptor tyrosine kinases (RTKs) to the Ras family of small G-proteins, which then stimulates the sequential activation of Raf serine/threonine kinases (MKKK), MEK (MKK), and ERK1/2 (MAP kinase) (12Marais R. Marshall C.J. Cancer Surv. 1996; 27: 101-125PubMed Google Scholar, 13Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (903) Google Scholar).Stimulation of cell proliferation and differentiation by FGF receptors acts via activation of the ERK pathway (14Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (566) Google Scholar, 15Noda M. Ko M. Ogura A. Liu D.G. Amano T. Takano T. Ikawa Y. Nature. 1985; 318: 73-75Crossref PubMed Scopus (348) Google Scholar). The small adaptor protein Grb2 has been shown to play an important role in linking RTK activation to the Ras/MAP kinase signaling pathway (16Schlessinger J. Curr. Opin. Genet. Dev. 1994; 4: 25-30Crossref PubMed Scopus (400) Google Scholar). Unlike most other RTKs, FGF receptors do not bind directly to Grb2 upon FGF stimulation. A novel lipid-anchored docking protein, FRS2, serves as a link between FGF receptor activation and the Ras/MAP kinase signaling pathway. FRS2 becomes tyrosine-phosphorylated upon FGF stimulation and associates with the Grb2·SOS complex to relay activation signals down the Ras/MAP kinase pathway (17Kouhara 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).Apart from the genetic evidence, relatively little is known about the biochemical mode of action of the various Spry isoforms. One study demonstrated that a considerable part of the conserved C-terminal portion was involved in translocating the protein from cytosol to membrane upon RTK stimulation of cells (18Lim J. Wong E.S. Ong S.H. Yusoff P. Low B.C. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Assuming this highly conserved sequence is involved in directing the cellular localization of the protein, it is reasonable to hypothesize that the C-terminal half of the protein is involved in binding proteins that are strategically placed to execute physiological functions such as inhibition of the Ras/MAP kinase pathway.Currently, genetic evidence derived entirely from studies inDrosophila indicates that the physiological role of dSpry is as an inhibitor of the Ras/MAP kinase pathway. However, the three studies that employed genetic approaches in Drosophila are not in agreement as to the mode and site of the inhibition induced by dSpry (1Hacohen 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, 2Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 3Reich A. Sapir A. Shilo B. Development. 1999; 126: 4139-4147Crossref PubMed Google Scholar). Mammalian Spry isoforms may have physiological activities that differ from those of dSpry and from each other. In this study, we asked several questions. In biochemical terms, do the various mammalian Spry isoforms inhibit the Ras/MAP kinase pathway? What features of the protein are required for the inhibition? Where does this inhibition take place?RESULTSBased on the observation in mammalian embryo development that the expression of Spry isoforms coincide with the expression of FGF rather than EGF (25Chambers D. Mason I. Mech. Dev. 2000; 91: 361-364Crossref PubMed Scopus (110) Google Scholar), we chose FGF-induced Ras/MAP kinase as our model system. Additionally we employed a cell culture system that best mimicked an inhibition of the pathway from the various in vivo systems reported: we reconstituted relatively long term stimulation of the FGF signaling pathway by overexpressing a wild type FGFR1 (flg) and observing the effects of overexpressed Spry isoforms at various assay points downstream with emphasis on the Ras/MAP kinase pathway.Full-length hSpry2 Is an Intracellular Inhibitor of the ERK Pathway but Not the JNK or p38 PathwaysFGFR1 was co-transfected into 293T cells with HA-tagged Spry isoforms and FLAG-tagged ERK2. Forty h after transfection, cells were lysed, and the lysates were precipitated with anti-FLAG. The immunoprecipitates and whole cell lysates were subjected to Western analysis using phospho-ERK1/2 (p42/44), ERK2, FGFR1, or HA antibodies to detect for phosphorylated ERK2 or the level of transfected proteins. The results in Fig.1A show that hSpry2 significantly inhibits the phosphorylation of ERK2, whereas neither mSpry1 nor mSpry4 has any significant inhibitory effect.Similar experiments were next performed, except that the ERK precipitates were subjected to in vitro kinase analysis as outlined under “Materials and Methods.” The data shown in Fig.1B further demonstrate a profound inhibition induced by hSpry2, which is consistent with the results in Fig. 1A.Previously we (18Lim J. Wong E.S. Ong S.H. Yusoff P. Low B.C. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) have shown that the C-terminal half of the hSpry2 protein was responsible for the growth factor-stimulated translocation of the protein to membrane ruffles. We next asked whether this region was able to inhibit the Ras/MAP kinase pathway when cells are stimulated with overexpressed FGFR1. Our results show that neither the C-terminal nor the N-terminal of hSpry2 was able to inhibit the Ras/MAP kinase pathway, which is in contrast to the inhibitory effects of full-length hSpry2. (Fig. 1C).From an earlier study in Drosophila, it was believed that dSpry exerts its inhibitory effect by competing exogenously with FGF (1Hacohen 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). Although subsequent evidence has demonstrated that dSpry2 operated on the inside of the plasma membrane (2Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), the issue has not been totally resolved. To investigate this issue in mammalian cells, we employed cytosolic FGFR1, which auto-activates to stimulate the Ras/MAP kinase pathway. Results from this experiment showed that FGFR1(cyto) stimulated phospho-ERK activity and that hSpry2 significantly inhibits this activation (Fig. 1D). Because there is no exogenous FGFR1 component, hSpry2 is exerting its inhibitory effect on the Ras/MAP kinase pathway endogenously.We next determined whether any of the other two generic MAP kinase pathways were inhibited by any of the Spry isoforms. Similar experiments to those outlined above were carried out, except that FLAG-tagged JNK1 or FLAG-tagged p38 was substituted for the ERK2 construct. The assays were calibrated using UV light (for JNK1) or Traf2 (for p38) stimulation as positive controls. The results shown in Fig. 1, E and F, indicate that none of the three Spry isoforms exert any effect on the phosphorylation of JNK1 or p38. It is apparent from the data above that hSpry2 is the only isoform that inhibits a generic MAP kinase pathway, and this inhibition is confined to the ERK pathway.hSpry2 Does Not Inhibit Either the Phosphorylation of FRS2 or the Associated Akt PathwayERK1/2 is somewhat distal from the receptor, and inhibition can potentially occur at various points along the pathway. To assess the site of inhibition of hSpry2, we decided to analyze key points along the canonical FGFR1 to ERK1/2 pathway.Current evidence indicates that FRS2 is the major “distribution center” of FGFR-derived signals in mammalian cells. It has been shown to link to the Ras/MAP kinase pathway via Grb2, SHP-2, and SOS and to the Akt pathway via Grb2, Gab-1, phosphoinositide 3-kinase, and phosphoinositide-dependent kinases (17Kouhara 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, 26Ong S.H. Hadari Y.R. Gotoh N. Guy G.R. Schlessinger J. Lax I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6074-6079Crossref PubMed Scopus (263) Google Scholar). We performed experiments to investigate whether any of the Spry isoforms affected FRS2α, either via stimulation-induced tyrosine phosphorylation or by direct binding. FRS2 cDNA was co-transfected with FGFR1 and hSpry2 constructs. The cell lysates were later subjected to separation on SDS-PAGE and subsequent Western analysis employing PY20 antibody to detect tyrosine phosphorylation of FRS2α. The data in Fig.2A show that hSpry2 has no effect on the tyrosine phosphorylation of FRS2α, which eliminates any effect of hSpry2 on the transactivation of FGFR1 as well as the ability of FGFR1 to phosphorylate FRS2α. As these experiments involve relatively long term stimulation of the FGF pathway, it is possible that hSpry2 could either act as an in vitrocompetitor of the receptor or cause the induction of an exogenous inhibitor, as has been suggested elsewhere (27Glienke J. Fenten G. Seemann M. Sturz A. Thierauch K.H. Mech. Dev. 2000; 96: 91-99Crossref PubMed Scopus (17) Google Scholar). The data shown preclude these possibilities as any impairment of receptor activity would present itself in the form of decreased tyrosine phosphorylation of its substrates, which is clearly not the case.Figure 2hSpry2 does not inhibit either the phosphorylation of FRS2α or the associated Akt pathway. hSpry2 has no effect on the tyrosine phosphorylation of FRS2α (A). 293T cells were transfected with wild type FRS2α, FGFR1, FLAG-Spry2, and empty vector constructs. Aliquots of the cell lysates were resolved by SDS-PAGE and tyrosine-phosphorylated FRS2α, and other related proteins were detected by immunoblotting (IB) with respective antibodies as indicated. Spry isoforms have no effect on the phosphoinositide 3-kinase pathway activated by FGFR1 (B). 293T cells were transfected with wild type Myc-Akt, FGFR1, Gab1, FLAG-tagged Sprys, and empty vector constructs. Anti-Myc immunoprecipitates were resolved by SDS-PAGE followed by Western blotting analysis to detect phospho-Akt and Akt using anti-phospho-Akt and anti-Akt, respectively. Other associated proteins in the cell lysates were detected using anti-Gab1, anti-FGFR1, and anti-FLAG. IP, immunoprecipitates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using GST-Spry2 pull-down experiments it was also shown that FRS2α does not bind to the Spry proteins (data not shown). This further indicates that the target of hSpry2 inhibition on the FGF signaling pathway is downstream of the docker protein.The activation of Akt via FRS2 and Gab1 represents a bifurcation from the Ras/MAP kinase pathway. Experiments were carried out using combinations of constructs for Myc-Akt, FGFR1, Gab1, and FLAG-tagged Spry isoforms to investigate whether hSpry2 elicited any effect on the Akt pathway. The cell lysates were subjected to precipitation with anti-Myc and assayed by Western analysis using antibodies against phospho-Akt. The results shown in Fig. 2B indicate that hSpry2 does not have any effect on the Akt pathway. Since hSpry2 did not have any effect at the level of receptor interaction with FRS2, our focus shifted to the next key point downstream; the possible effects on Ras activity.hSpry2 Does Not Inhibit the Activity of RasThe activation status of Ras was measured as described under “Materials and Methods.” The main component of the assay was a recombinant protein derived from Raf that contains a domain capable of binding only to GTP-bound Ras (Raf-1 Ras binding domain). 293T cells were transfected with various combinations of constructs for FGFR1 and Spry isoforms. The results shown in Fig. 3indicate that although transfected FGFR1 significantly enhanced the level of GTP-Ras, hSpry2 co-transfected into cells had no significant effect on these levels. Equal loading of whole cell lysates showed an inhibition of endogenous phospho-ERK by hSpry2, as demonstrated in previous experiments. Similar results were obtained in three such experiments. Accumulated data therefore indicated that hSpry2 did not inhibit the Ras/MAP kinase pathway between the point of receptor activation and the activation of Ras. These data also rule out hSpry2's sequestration of Grb2 from SOS as a likely inhibitory mechanism. It was therefore necessary to assay the effect of hSpry2 on elements of the Ras/MAP kinase pathway downstream of Ras. It is apparent that there is some inhibition by mSpry1 and mSpry4, but it is very low compared with hSpry2.Figure 3Sprys do not inhibit FGFR1 activated Ras. 293T cells were transfected with FGFR1 FLAG-tagged Sprys (S1, S2, and S4) or empty vector constructs as indicated (C). Cell lysates were incubated with GST-Raf-1 Ras binding domain (RBD) agarose-conjugated beads. Bound proteins were denatured by boiling in reducing sample buffer and then resolved by SDS-PAGE followed by Western blotting analysis to detect Ras-GTP using a monoclonal anti-Ras. Aliquots of the same cell lysates were resolved by SDS-PAGE, and phospho-ERK1/2 and other related proteins were detected by immunoblotting (IB) as indicated. (S1, Sprouty1; S2, Sprouty2;S4, Sprouty4; PD, pull-down.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)hSpry2 Inhibits ERK Phosphorylation Downstream of Ras and Upstream of MEK and ERKExperiments were carried out to locate the position of MAP kinase inhibition along the pathway from Ras to ERK. This pathway essentially involves three proteins: the G-protein Ras, which activates the protein kinase Raf; Raf which activates Mek; and Mek, which in turn activates ERK.We took advantage of mutations in Ras, Raf, and Mek that cause an auto-activation of each of these respective proteins. Constructs were transfected into 293T cells along with FLAG-tagged ERK2 and HA-tagged Spry isoforms. In each of these experiments, the cells were lysed, and the lysates were precipitated with anti-FLAG beads and analyzed by Western blotting using phospho-ERK1/2 antibody to ascertain the activation status of ERK (MAP kinase). The data from Fig.4A show that hSpry2 can inhibit the phosphorylation (activation) of ERK2 downstream of active Ras (V12). A distinct inhibition of phosphorylated ERK2 was also observed in the mSpry1-transfected cells; however, the degree of inhibition by hSpry2 was more profound, and we subsequently focused more on the effects of this isoform. In addition, we demonstrated that the inhibition of activated ERK2 stimulated through the Ras mutant requires the full-length hSpry2 protein since neither the C- or N-terminal half was able to inhibit this activity (Fig.4B). Contrary to the inhibition of activated Ras-generated signals, full-length hSpry2 did not cause any detectable inhibition of ERK2 phosphorylation when the constitutively active mutants of Raf (RafY340D) or Mek (MekS222D/S226D), respectively, were employed to activate the pathway (Fig. 4, C and D).Figure 4hSpry2 inhibits ERK phosphorylation downstream of Ras and upstream of MEK. 293T cells were transfected with the constitutively active Ras (RasV12) mutant, wild type FLAG-ERK2, HA-Sprys, or empty vector constructs as indicated (A). Anti-FLAG immunoprecipitates (IP) were resolved by SDS-PAGE followed by Western blotting analysis to detect phospho-ERK2 and ERK2 levels using anti-phospho-ERK1/2 and anti-ERK2, respectively. Ras and Spry proteins were detected using anti-Ras and anti-HA in the total cell lysates, respectively. IB, immunoblots. Inhibition of phosphorylated ERK through active Ras requires the full-length hSpry2 (B). 293T cells were transfected with wild type FLAG-ERK2, constitutively active Ras (RasV12), HA-tagged full-length Spry2, the N- or C-terminal half of hSpry2, and empty vector constructs. Anti-FLAG immunoprecipitates were resolved by Western blotting analysis to detect phospho-ERK2 and ERK2 using anti-phospho-ERK1/2 (phospho-p42/44) and anti-ERK2, respectively. Other associated proteins in the cell lysates were detected using anti-Ras and anti-HA, respectively. Sprys do not inhibit ERK1/2 activation by the Raf (Y340D) mutant (C). 293T cells were transfected with wild type FLAG-ERK2, constitutively active mutants of Raf (RafY340D), HA-Sprys, or empty vector constructs as indicated. Anti-FLAG immunoprecipitates were resolved by SDS-PAGE followed by Western blotting analysis to detect phospho-ERK2 and ERK2 using anti-phospho-ERK1/2 and anti-ERK2, respectively. Raf and Spry proteins were detected in the cell lysates using anti-Raf and anti-HA antibodies, respectively. hSpry2 acts on the Ras/MAP kinase pathway upstream of MEK (D). 293T cells were transfected with wild type FLAG-ERK2, constitutively active mutants of Mek (MekS222D/S226D), HA-Sprys, or empty vector constructs as indicated. Anti-FLAG immunoprecipitates were resolved by SDS-PAGE followed by Western blotting analysis to detect phospho-ERK2 and ERK2 proteins using anti-phospho-ERK1/2 and anti-ERK2, respectively. MEK and Spry proteins were detected in the cell lysates using anti-HA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The above data indicate that hSpry2 is exerting its inhibitory effect at the level of activation of Raf. Raf has a complex mechanism of activation, and there currently appears to be some unidentified factors, especially kinases that may be involved. It would also appear from these data that hSpry2 should inhibit the MAP kinase pathway irrespective of what agonist stimulates it because the active Ras mutant used in this study is a generic stimulant that is independent of upstream pathways.hSpry2 Inhibits ERK Activation Induced by β-2 Adrenergic Receptor StimulationA wide range of agonists activates the Ras/MAP kinase pathway. Sprouty has been shown to inhibit signaling pathways induced by receptor tyrosine kinases. Based on our results, we were interested in investigating the effects of hSpry when a non-RTK (for instance the isoprenaline activation of β2 adrenergic receptors (β2ARs)), was used to stimulate the Ras/MAP kinase pathway. To address this, 293T cells were transfected with constructs for β2AR, HA-tagged hSpry2, and FLAG-tagged ERK2. Forty-eight h post-transfection, the cells were stimulated with isoprenaline (100 μm) for various times, the cells were lysed, and the lysates were subjected to Western analysis using anti-phospho ERK1/2 to detect the level of ERK phosphorylation. The data shown in Fig. 5demonstrate that ERK2 phosphorylation was elevated 5 min after isoprenaline addition and that this level was sustained for the next 25 min at least. hSpry2 substantially inhibited ERK2 phosphorylation to the degree that any level of phosphorylation in the data shown was only evident at 5 min. It has been reported that isoprenaline activation of β2AR can activate EGFR by “cross-talk” and that this activation can contribute to the subsequent activation of the Ras/MAP kinase pathway (28Maudsley S. Pierce K.L. Zamah A.M. Miller W.E. Ahn S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2000; 275: 9572-9580Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The point illustrated by the above experiment is that it appears hSpry2 will inhibit the Ras/MAP kinase pathway downstream of activated Ras in a manner that is not dependent on the mode of pathway activation.Figure 5hSpry2 inhibits ERK activation induced by β2 adrenergic receptor stimulation.293T cells transfected with HA-hSpry2 and β2 adrenergic receptor were serum-depleted overnight and subsequently stimulated with isoprenaline (100 μm) for various times. Cells were lysed and immunoprecipitated with FLAG antibody. The anti-FLAG immunoprecipitates (IP) were resolved by Western blotting analysis to detect phospho-ERK2 and ERK2 using anti-phospho-ERK1/2 (phospho-p42/44) and anti-ERK2, respectively. Other associated proteins in the cell lysates were detected using anti-HA. IB, immunoblots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The accumulated data indicate that hSpry2 is the predominant inhibitor of the Ras/MAP kinase and that its inhibitory effect is exerted at the level of Raf activation. In this case, hSpry2 should inhibit the activation of Raf.hSpry2 Inhibits the Activation of RafThe effect of the various Spry isoforms on the status of Raf stimulation downstream of active FGFR1 was analyzed. FGFR1 was transiently expressed in 293T cells, and Raf kinase activity was measured by a coupled assay using GST-MEK, GST-ERK, and GST-Elk or myelin basic protein as sequential substrates. The assay measures the phosphotransferase activity in anin vitro kinase cascade reaction initiated in the immunocomplex by active Raf-1. The results show that FGFR1 significantly stimulated the Raf kinase activity in comparison with the vector control. When the cells were co-transfected with the various Spry isoforms, it can be seen that hSpry2 profoundly reduced the level of Raf kinase activity, whereas mSpry1 and mSpry4 caused lesser inhibition (Fig. 6A). A parallel inhibition of phosphorylated ERK2 was observed with hSpry2 overexpression, using the same cell lysates and Western blotting detection (Fig. 6B). In essence, hSpry2, in comparison with mSpry1 and mSpry4, causes a more profound inhibition of the Ras/MAP kinase pathway, and this down-regulation occurs at the level of Raf kinase activation.Figure 6Effect of Sprys on activated Raf-1 byFGFR1. 293T cells were transfected with empty vector (V), FGFR1 (C), or FGFR1 and FLAG-Sprys (S1, S2, and S4) constructs as indicated. Raf-1 was immunoprecipitated from lysates, and Raf activity was assessed by an in vitro coupled kinase assay in which recombinant MEK, ERK, and Elk-1 were used. Raf-1 activity was determined quantitatively by scintillation counting of the P81 phosphocellulose squares spotted with the assay product. Background c" @default.
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• W2070655784 date "2002-02-01" @default.
• W2070655784 modified "2023-10-12" @default.
• W2070655784 title "Sprouty2 Inhibits the Ras/MAP Kinase Pathway by Inhibiting the Activation of Raf" @default.
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• W2070655784 doi "https://doi.org/10.1074/jbc.m108368200" @default.
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