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• W2003289800 abstract "Rho GTPases are molecular “switches” that cycle between “on” (GTP-bound) and “off” (GDP-bound) states and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood. Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors and that this event is critical for the regulated activation of the GEF. These findings uncover a novel layer of complexity in the physiological regulation of this protein. Rho GTPases are molecular “switches” that cycle between “on” (GTP-bound) and “off” (GDP-bound) states and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood. Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors and that this event is critical for the regulated activation of the GEF. These findings uncover a novel layer of complexity in the physiological regulation of this protein. The Dbl family of guanine nucleotide exchange factors (GEFs) 2The abbreviations used are:GEFguanine nucleotide exchange factorEGFREGF receptorPP24-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. comprises >80 members that facilitate the activation of the small GTPases Rac1, Cdc42, and/or RhoA. Dbl was the first identified mammalian GEF and as such is considered to be the prototypic member of the family (1Srivastava S.K. Wheelock R.H. Aaronson S.A. Eva A. Identification of the protein encoded by the human diffuse B-cell lymphoma (dbl) oncogene.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 8868-8872Crossref PubMed Scopus (33) Google Scholar). Two highly conserved domains characterize Dbl family GEFs: a unique Dbl homology domain and a pleckstrin homology domain. The Dbl homology domain is the minimal unit necessary for GEF activity that binds the substrate GTPase and catalyzes the exchange of GDP for GTP in its binding pocket (2Hart M.J. Eva A. Evans T. Aaronson S.A. Cerione R.A. Catalysis of guanine nucleotide exchange on the Cdc42Hs protein by the dbl oncogene product.Nature. 1991; 354: 311-314Crossref PubMed Scopus (336) Google Scholar). Following GEF-mediated activation, the bound GTP is hydrolyzed, and the GTPase returns to its quiescent state. The pleckstrin homology domain is thought to mediate proper localization of the GEF to specific subcellular compartments (3Haslam R.J. Koide H.B. Hemmings B.A. Pleckstrin domain homology.Nature. 1993; 363: 309-310Crossref PubMed Scopus (387) Google Scholar, 4Russo C. Gao Y. Mancini P. Vanni C. Porotto M. Falasca M. Torrisi M.R. Zheng Y. Eva A. Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain.J. Biol. Chem. 2001; 276: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 5Zheng Y. Zangrilli D. Cerione R.A. Eva A. The pleckstrin homology domain mediates transformation by oncogenic Dbl through specific membrane targeting.J. Biol. Chem. 1996; 271: 19017-19020Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In response to growth factor stimulation, Dbl-mediated GTPase signaling regulates numerous cellular activities such as cytoskeletal rearrangements, gene expression, and vesicular trafficking, thereby promoting cell proliferation (6Cerione R.A. Zheng Y. The Dbl family of oncogenes.Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar, 7Zheng Y. Dbl family guanine nucleotide exchange factors.Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 8Rossman K.L. Der C.J. Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.Nat. Rev. Mol. Cell Biol. 2005; 6: 167-180Crossref PubMed Scopus (1314) Google Scholar, 9Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Dbl family proteins.Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar, 10Erickson J.W. Cerione R.A. Multiple roles for Cdc42 in cell regulation.Curr. Opin. Cell Biol. 2001; 13: 153-157Crossref PubMed Scopus (150) Google Scholar). guanine nucleotide exchange factor EGF receptor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Because GTPases control multiple and diverse aspects of cellular physiology, proper regulation of GEF activities is crucial for normal cellular function. Indeed, mutations that cause GEF overexpression or gain of function result in persistent activation of downstream pathways and lead to aberrant cell growth that manifest in developmental disorders and cancers. For example, mutations in Fgd1 cause the X-linked developmental disorder, Aarskog-Scott syndrome (11Pasteris N.G. Cadle A. Logie L.J. Porteous M.E. Schwartz C.E. Stevenson R.E. Glover T.W. Wilroy R.S. Gorski J.L. Isolation and characterization of the faciogenital dysplasia (Aarskog-Scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor.Cell. 1994; 79: 669-678Abstract Full Text PDF PubMed Scopus (269) Google Scholar), and chromosomal rearrangements of the Bcr gene contribute to leukemogenesis (12Groffen J. Stephenson J.R. Heisterkamp N. de Klein A. Bartram C.R. Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22.Cell. 1984; 36: 93-99Abstract Full Text PDF PubMed Scopus (1248) Google Scholar). Mutations in Plekhg4 are associated with heritable autosomal spinocerebellar ataxia (13Ishikawa K. Mizusawa H. On autosomal dominant cerebellar ataxia (ADCA) other than polyglutamine diseases, with special reference to chromosome 16q22.1-linked ADCA.Neuropathology. 2006; 26: 352-360Crossref PubMed Scopus (12) Google Scholar, 14Ishikawa K. Toru S. Tsunemi T. Li M. Kobayashi K. Yokota T. Amino T. Owada K. Fujigasaki H. Sakamoto M. Tomimitsu H. Takashima M. Kumagai J. Noguchi Y. Kawashima Y. Ohkoshi N. Ishida G. Gomyoda M. Yoshida M. Hashizume Y. Saito Y. Murayama S. Yamanouchi H. Mizutani T. Kondo I. Toda T. Mizusawa H. An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5′ untranslated region of the gene encoding a protein with spectrin repeat and rho guanine-nucleotide exchange-factor domains.Am. J. Hum. Genet. 2005; 77: 280-296Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Overexpression of P-rex1, Ect2, and Tiam1 has been noted in cancers of the breast, lung, and colon, respectively (15Hynes N.E. Gattelli A. P-Rex1, a guanine exchange factor that is overexpressed in breast cancer, is a convergence node for ErbB and CXCR4 signaling.Mol. Cell. 2011; 41: 5-7Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 16Justilien, V., Jameison, L., Der, C. J., Rossman, K. L., Fields, A. P., Oncogenic activity of Ect2 is regulated through protein kinase C ι-mediated phosphorylation. J. Biol. Chem. 286, 8149–8157Google Scholar, 17Fields, A. P., Justilien, V., The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer. Adv. Enzyme Regul. 50, 190–200Google Scholar, 18Cottonham, C. L., Kaneko, S., Xu, L., miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells. J. Biol. Chem. 285, 35293–35302Google Scholar, 19Minard M.E. Ellis L.M. Gallick G.E. Tiam1 regulates cell adhesion, migration and apoptosis in colon tumor cells.Clin. Exp. Metastasis. 2006; 23: 301-313Crossref PubMed Scopus (87) Google Scholar, 20Minard M.E. Herynk M.H. Collard J.G. Gallick G.E. The guanine nucleotide exchange factor Tiam1 increases colon carcinoma growth at metastatic sites in an orthotopic nude mouse model.Oncogene. 2005; 24: 2568-2573Crossref PubMed Scopus (59) Google Scholar), and overexpression of Dbl was reported in sarcomas and tumors of neuroectodermal origins (21Vecchio G. Cavazzana A.O. Triche T.J. Ron D. Reynolds C.P. Eva A. Expression of the dbl proto-oncogene in Ewing's sarcomas.Oncogene. 1989; 4: 897-900PubMed Google Scholar, 22Colucci-D'Amato G.L. De Franciscis V. Rosati R. Mauro A. Bulfone A. Eva A. Vecchio G. Preferential expression of the dbl proto-oncogene in some neuroectodermal tumors.J. Neurosurg. Sci. 1990; 34: 187-188PubMed Google Scholar, 23Navarro S. Pellín A. Noguera R. Díaz M.P. Tsokos M. Triche T.J. Llombart-Bosch A. dbl oncogene expression in childhood tumors and tumor cell lines.Diagn. Mol. Pathol. 1993; 2: 158-162Crossref PubMed Scopus (3) Google Scholar, 24Velasco J.A. Ramsamooj P. Thraves P.J. Eva A. Dritschilo A. Notario V. Co-regulated expression of dbl and poly(ADP-ribose) polymerase in Ewing's sarcoma cells and dbl-transformed NIH3T3 fibroblasts.Oncogene. 1995; 10: 2253-2258PubMed Google Scholar). As our appreciation for the involvement of Dbl family GEFs in disease grows, the need for understanding the mechanisms that regulate these proteins increases. The upstream events that regulate the GEF activity of Dbl are poorly understood. Dbl is thought to exist in an inhibited state, which is activated by growth factor receptors; however, the molecular mechanisms that underlie the inhibited state and its release are incompletely understood. Some studies proposed an intramolecular autoinhibited state brought about by interactions between the amino and carboxyl termini of Dbl (25Bi F. Debreceni B. Zhu K. Salani B. Eva A. Zheng Y. Autoinhibition mechanism of proto-Dbl.Mol. Cell Biol. 2001; 21: 1463-1474Crossref PubMed Scopus (68) Google Scholar). Other studies demonstrated that association with the molecular chaperones Hsc70 and Hsp90 regulate Dbl activity by controlling its degradation by the proteasome (26Kamynina E. Kauppinen K. Duan F. Muakkassa N. Manor D. Regulation of proto-oncogenic dbl by chaperone-controlled, ubiquitin-mediated degradation.Mol. Cell Biol. 2007; 27 (PMC1820456): 1809-1822Crossref PubMed Scopus (32) Google Scholar, 27Kauppinen K.P. Duan F. Wels J.I. Manor D. Regulation of the Dbl proto-oncogene by heat shock cognate protein 70 (Hsc70).J. Biol. Chem. 2005; 280: 21638-21644Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The mechanisms by which growth factor stimulation leads to release of Dbl inhibition are completely unknown. In this study, we aimed to understand the upstream events that relieve Dbl autoinhibition and stimulate its GEF activity. We show that a key feature of the activation cycle of Dbl involves reversible, growth factor-sensitive, tyrosine phosphorylation. COS7 cells and HEK293T cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in 5% CO2 at 37 °C. NIH3T3 cells were cultured in DMEM supplemented with 10% calf serum (Hyclone). Parental and EGFR-expressing CHO cells were a generous gift from Dr. Cathleen Carlin, Case Western Reserve University School of Medicine and were cultured in minimum essential mediumα supplemented with 10% FBS (Hyclone). Dbl constructs encoded the reading frame encoding the full-length protein (residues 1–925) in the pCMV6 vector, or the pCEFL-GST vector (for focus formation experiments). Recombinant GTPases were expressed in Escherichia coli as glutathione S-transferase fusions and purified as described previously (28Leonard D.A. Evans T. Hart M. Cerione R.A. Manor D. Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.Biochemistry. 1994; 33: 12323-12328Crossref PubMed Scopus (69) Google Scholar). Grb2 cDNA (HA-tagged in the pCGN vector) was a generous gift from Dr. Dafna Bar-Sagi, New York University School of Medicine. All transfections were done using PolyFect (Qiagen). EGF (Sigma) was dissolved in serum-free DMEM and used at a final concentration of 50 nm. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, EMD Millipore) was dissolved in dimethyl sulfoxide and used at a final concentration of 1 or 10 μm, as was Iressa (gefitnib, Selleck Biochemicals). The phosphatase inhibitor-activated sodium orthovanadate (Sigma) was added during the last 10 min to a final concentration of 1 mm. Cells were lysed in 1 ml of 20 mm Hepes, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1% Igepal, 20 mm NaF, 20 mm β-glycerophosphate, 1 mm vanadate, 200 μm phenylmethylsulfonyl fluoride, and 10 μg/ml each of leupeptin and aprotinin. Lysates were spun at 14,000 rpm for 20 min, and cleared supernatant was combined with anti-Dbl antibody (Santa Cruz Biotechnology, sc-89, 1:100 dilution) or anti-HA antibody (Covance, MMS-101R, 1:100). The lysate-antibody mixture was mixed at 4 °C for 1 h before the addition of 50 μl of protein A beads (Millipore) and mixing at 4 °C for 2.5 h. Beads were washed three times with lysis buffer and spun at 3,500 rpm for 3 min. Precipitated beads were boiled in 2.5× Lamelli buffer, and the supernatant was resolved by 8–12% SDS-PAGE. Tyrosine phosphorylation was visualized using anti-phosphotyrosine antibody (4G10-platinum, Millipore, 1:1000 dilution). Whole brain extracts from 10-day-old mice were homogenized manually in 1 ml of 150 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 150 mm NaCl, 25 mm Tris-HCl (pH 8.0), 0.25 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of leupeptin and aprotinin. The homogenate was cleared by centrifugation at 14,000 rpm for 45 min at 4 °C, followed by immunoprecipitation with Dbl antibody as described above. Site-directed mutagenesis was performed according to the manufacturer's instructions (QuikChange XL II, Agilent). Five hours prior to lysis, cells were switched to MEMα containing 0.5% FBS. After 3 h, cells were placed on ice for 2 h and treated as indicated (EGF, Iressa, or PP2). Cells were then incubated at 37 °C for 15 min, lysed, and subjected to immunoprecipitations as described above. COS7 cells were co-transfected with HA-Grb2 and Dbl constructs, harvested in 50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 10 mm pyrophosphate, 10 mm glycerophosphate, 50 mm NaF, 1 mm vanadate, 200 μm phenylmethylsulfonyl fluoride, and 10 μg/ml each leupeptin and aprotinin, and centrifuged at 14,000 rpm for 20 min. Association was visualized by anti-HA immunoblotting of anti-Dbl immunoprecipitates. These were designed based on the established high affinity of GEFs to their nucleotide-free form of their cognate GTPases (29Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product.J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar, 30Bourne H.R. Sanders D.A. McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions.Nature. 1990; 348: 125-132Crossref PubMed Scopus (1836) Google Scholar, 31Bourne H.R. Sanders D.A. McCormick F. The GTPase superfamily: conserved structure and molecular mechanism.Nature. 1991; 349: 117-127Crossref PubMed Scopus (2684) Google Scholar). Wild-type GTPases and their respective nucleotide-free mutants (Cdc42(T17N) or RhoA(T19N)) were expressed in E. coli as GST fusions and purified as described previously (32Wu W.J. Leonard D.A. A-Cerione R. Manor D. Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region.J. Biol. Chem. 1997; 272: 26153-26158Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The purified GTPases were immobilized on glutathione agarose and washed extensively with cell lysis buffer supplemented with 50 μm GDP, 10 mm MgCl2 (wild-type GTPase), or 10 mm EDTA (Asn17/Asn19 GTPase). Dbl-transfected cells were lysed in the same lysis buffer, incubated with immobilized GTPases at 4 °C for 2 h, and washed three times in the respective buffers, prior to SDS-PAGE and anti-Dbl immunoblotting. To measure GEF activity, the fraction of endogenous GTPase that is in the active (GTP-bound) state was determined using an established selective precipitation approach (33Bagrodia S. Dérijard B. Davis R.J. Cerione R.A. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation.J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 34Ren X.D. Schwartz M.A. Determination of GTP loading on Rho.Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar, 35Taylor S.J. Shalloway D. Cell cycle-dependent activation of Ras.Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Briefly, HEK293T cells were transfected with Dbl cDNA, serum-starved for 31 h, and lysed in 20 mm HEPES (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 10% glycerol, 10 mm MgCl2, 1 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of leupeptin and aprotinin. Lysates were spun at 14,000 rpm for 20 min, and cleared supernatant was combined with ∼20 μg of immobilized GST-fused GTPase-binding domain of rhotekin and rotated at 4 °C for 2 h (34Ren X.D. Schwartz M.A. Determination of GTP loading on Rho.Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar). Beads were washed three times in lysis buffer and resolved on SDS-PAGE. The amount of activated (bead-associated) GTPase was visualized with anti-RhoA (Cytoskeleton, 1:200 dilution) immunoblotting and compared with the total GTPase levels as determined by anti-RhoA immunoblotting of whole cell lysates. NIH3T3 cells were plated in triplicate in a 24-well plate and co-transfected with the NFκB response element fused to the luciferase reporter construct (pGL3B-HIV1-luc), the β-galactosidase-expressing pCH110 (Pharmacia), and Dbl cDNA. Thirty-six hours after transfection, cells were washed in serum-free medium and starved for 17 h prior to harvesting cell lysates and measuring luciferase and β-galactosidase activities. Luciferase expression was normalized to β-galactosidase activity to account for differences in transfection efficiency. The indicated constructs were used to transfect subconfluent NIH3T3 cells in 10-cm plates. After 48 h, media was replaced, and cells were refed every other day. Approximately 2 weeks after transfection, cells were fixed with methanol and stained with crystal violet, and foci were manually scored. NIH3T3 cells were seeded on glass coverslips and transfected with the indicated constructs (opr empty pCMV6 vector as control) using PolyFect according to the manufacturer's instructions (Qiagen). Thirty-one hours post-transfection, cells were serum-starved for 17 h, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with the indicated reagents, and imaged using the Leica TCS SP2 microscope. Dbl proteins were visualized using Santa Cruz Biotechnology sc-89 antibody and a secondary Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) antibody; the actin cytoskeleton was visualized with Texas Red-conjugated phalloidin (Invitrogen). In a search for post-translational events that may regulate the GEF activity of Dbl, we found that anti-Dbl immunoprecipitates give a strong signal on anti-phosphotyrosine immunoblots. Importantly, the signal was only visible in the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (Fig. 1A). The physiological relevance of this finding is evident by the observation that tyrosine phosphorylation of Dbl occurs in intact tissue where the protein is expressed, such as mouse brain (Fig. 1B). Moreover, we found that when serum-deprived COS7 cells are treated with serum, Dbl phosphorylation transiently increases after 2–5 min and then tapers off (Fig. 1C). These observations show that Dbl is phosphorylated on tyrosine residue(s) and that this modification is a regulated event, dependent on growth factors. To begin to delineate the upstream events that regulate Dbl phosphorylation, we used pharmacological inhibitors of two major intracellular tyrosine kinases: Iressa (gefitinib), which inhibits the EGF receptor tyrosine kinase, and the inhibitor of Src family tyrosine kinases, PP2. To confirm these findings we examined whether expression of the EGFR kinase affects the phosphorylation of Dbl. In CHO cells that stably overexpress the EGFR kinase, stimulation with EGF caused a marked phosphorylation of Dbl (Fig. 1D). Moreover, modification was inhibited by treatment with either Iressa or PP2. Importantly, Dbl expression, but not phosphorylation, was observed in the parental CHO cell line, which does not express EGFR (Fig. 1D). Taken together, these findings indicate that phosphorylation of Dbl is mediated by mitogenic stimulation through the activated EGF receptor and a Src family kinase. Furthermore, the data suggest that in this pathway, the Src family kinase is downstream of EGFR. We utilized a bioinformatics approach to identify the tyrosine residue(s) that are phosphorylated in Dbl. The NetPhos2.0 algorithm (36Blom N. Sicheritz-Pontén T. Gupta R. Gammeltoft S. Brunak S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence.Proteomics. 2004; 4: 1633-1649Crossref PubMed Scopus (1486) Google Scholar) identified seven residues in Dbl that reside within tyrosine phosphorylation consensus sequences. These putative phosphorylation sites are dispersed throughout the different domains of the protein (Tyr217, Tyr510, Tyr553, Tyr749, Tyr780, Tyr787, and Tyr904; see Fig. 2A). We focused our attention to Tyr510, due to its position in a high-scoring putative phosphorylation sequence in the catalytic Dbl homology domain. Using site-directed mutagenesis, we substituted Tyr510 of Dbl to a phenylalanine and observed a dramatic decrease (∼80%) in the tyrosine phosphorylation intensity of the protein (Fig. 2B). The fact that some tyrosine phosphorylation persists in the Y510F mutant indicates that sites of tyrosine phosphorylation other than Tyr510 exist in the protein. In attempting to identify such residues, we found that mutation of Tyr787 did not affect the residual phosphorylation signal observed in Dbl(Y510F) (data not shown).FIGURE 2Tyr510 is an important site of phosphorylation in Dbl. A, domain structure of the Dbl protein. Of the 29 tyrosines of Dbl, the top-scoring putative phosphorylation sites are indicated, as well as the immediate sequence surrounding Tyr510. B, tyrosine phosphorylation of Dbl(Y510F) was examined as in Fig. 1A. Data are representative of three independent experiments. PY, phosphotyrosine; IB, immunoblot; DH, Dbl homology domain; PH, pleckstrin homology domain; SPEC, spectrin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To understand the molecular consequences of tyrosine phosphorylation in Dbl, we examined its ability to associate with established effectors. Grb2 is an adapter molecule that contains an Src homology 2 domain that binds phosphorylated tyrosine residues and an Src homology 3 domain that binds proline-rich sequences (37Downward J. The GRB2/Sem-5 adaptor protein.FEBS Lett. 1994; 338: 113-117Crossref PubMed Scopus (162) Google Scholar). Due to this dual-recognition capacity, Grb2 often functions as a “bridging” adapter, allowing it to recruit multiple proteins to their cellular site of activation, as seen in the activation of Ras, where Grb2 recruits Son of Sevenless to the tyrosine-phosphorylated EGF receptor (38Chardin P. Camonis J.H. Gale N.W. van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.Science. 1993; 260: 1338-1343Crossref PubMed Scopus (657) Google Scholar, 39Gale N.W. Kaplan S. Lowenstein E.J. Schlessinger J. Bar-Sagi D. Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras.Nature. 1993; 363: 88-92Crossref PubMed Scopus (453) Google Scholar). Because Grb2 was recently reported to be associated with Dbl (40Blajecka, K., Marinov, M., Leitner, L., Uth, K., Posern, G., Arcaro, A., Phosphoinositide 3-kinase C2β regulates RhoA and the actin cytoskeleton through an interaction with Dbl. PLoS One 7, e44945Google Scholar), we sought to evaluate the role of Tyr510 phosphorylation in this interaction. As seen in Fig. 3A, substitution of the Tyr510 of Dbl with a non-phosphorylatable phenylalanine residue greatly diminished Grb2 binding. Furthermore, the association between Grb2 and Dbl required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (Fig. 3B). Together, these data strongly suggest that phosphorylation of the Tyr510 of Dbl is critical for its binding to Grb2. Next, we asked whether tyrosine phosphorylation of Dbl is necessary for association of the GEF with its substrate GTPase. Cells expressing Dbl (or its phospho-defective mutant) were lysed and precipitated with immobilized recombinant GTPases that have been purified in either the GDP-bound form (GST-fused wild-type GTPases) or the nucleotide-free form (GST-RhoA(T19N) or GST-Cdc42(T17N) mutants). The rationale for this design is based on the notion that the highest affinity of GEFs to their GTPase is observed in the nucleotide-free form of the latter (29Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product.J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar, 30Bourne H.R. Sanders D.A. McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions.Nature. 1990; 348: 125-132Crossref PubMed Scopus (1836) Google Scholar, 31Bourne H.R. Sanders D.A. McCormick F. The GTPase superfamily: conserved structure and molecular mechanism.Nature. 1991; 349: 117-127Crossref PubMed Scopus (2684) Google Scholar). As expected, we observed a strong preference in the association of Dbl with the nucleotide-free forms of RhoA and Cdc42, as compared with the GDP-bound GTPases (compare lanes 1 and 2 in Fig. 4, A and B). Importantly, we observed that the affinity of the nucleotide-free GTPase to the GEF is dramatically reduced when phosphorylation of Tyr510 of Dbl is abolished by mutation to phenylalanine (Fig. 4, A and B). These findings indicate that that phosphorylation of Tyr510 is required for Dbl to be able to bind substrate Rho GTPases. To address the role of Tyr510 phosphorylation in Dbl activity as a GEF, we examined its ability to stimulate GTP binding by a substrate GTPase. Because both Cdc42 and Rac1 are phosphorylated on Tyr64 (41Tu S. Wu W.J. Wang J. Cerione R.A. Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase.J. Biol. Chem. 2003; 278: 49293-49300Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), 3M. Gupta, X. Qi, V. Thakur, and D. Manor, unpublished results. whereas RhoA is not (Fig. 5A), we chose to focus on the latter GTPase. This allowed us to evaluate the effect of kinase inhibitors on Dbl actions without complication arising from the effect of the inhibitors on the substrate GTPase. We treated Dbl-transfected cells with Iressa and measured the GEF activity of Dbl by selectively precipitating GTP-bound form of RhoA ((33Bagrodia S. Dérijard B. Davis R.J. Cerione R.A. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation.J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 34Ren X.D. Schwartz M.A. Determination of GTP loading on Rho.Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar, 35Taylor S.J. Shalloway D. Cell cycle-dependent activation of Ras.Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar); see “Materials and Methods”). We found that treatment with Iressa all but abolished the ability of Dbl to activate RhoA (Fig. 5B), indicating that EGFR-mediated tyrosine phosphorylation of Dbl is essential for its activity as a GEF. Importantly, substitution of Tyr510 to phenylalanine abolished the GEF activity of Dbl (Fig. 5C). Next, we examined the role of Tyr510 phosphorylation on further downstream signaling events. We found that the Y510F mutant of Dbl abolished transcriptional activation of NFkB (Fig. 6A), a downstream effector of RhoA, Rac1, and Cdc42 (42Cammarano M.S. Minden A. Dbl and the Rho GTPases activate NF κB by I κB kinase (IKK)-dependent and IKK-independent pathways.J. Biol. Chem. 2001; 276: 25876-25882Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 43Montaner S. Perona R. Saniger L. Lacal J.C. Multiple signalling pathways lead to the activation of the nuclear factor κB by the Rho family of GTPases.J. Biol. Chem. 1998; 273: 12779-12785Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 44Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Dependence of Dbl and Dbs transformation on MEK and NF-κB activation.Mol. Cell Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar). These results further confirm that tyrosine phosphorylation of Dbl is necessary to activate the Rho family small GTPases. Lastly, we examined at the role of Tyr510 phosphorylation on the signature biological activity of Dbl: transformation of cultu" @default.
• W2003289800 created "2016-06-24" @default.
• W2003289800 creator A5013520979 @default.
• W2003289800 creator A5032924715 @default.
• W2003289800 creator A5038118493 @default.
• W2003289800 creator A5086653443 @default.
• W2003289800 date "2014-06-01" @default.
• W2003289800 modified "2023-10-16" @default.
• W2003289800 title "Tyrosine Phosphorylation of Dbl Regulates GTPase Signaling" @default.
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