The Death Effector Domain of PEA-15 Is Involved in Its Regulation of Integrin Activation
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.33897
ISSN1083-351X
AutoresJoe W. Ramos, Thomas K. Kojima, Paul E. Hughes, Csilla A. Fenczik, Mark H. Ginsberg,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoIncreased integrin ligand binding affinity (activation) is triggered by intracellular signaling events. A Ras-initiated mitogen-activated protein kinase pathway suppresses integrin activation in fibroblasts. We used expression cloning to isolate cDNAs that prevent Ras suppression of integrin activation. Here, we report that PEA-15, a small death effector domain (DED)-containing protein, blocks Ras suppression. PEA-15 does not block the capacity of Ras to activate the ERK mitogen-activated protein kinase pathway. Instead, it inhibits suppression via a pathway blocked by a dominant-negative form of the distinct small GTPase, R-Ras. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to reverse suppression of integrin activation. Thus, certain DED-containing proteins can regulate integrin activation as opposed to apoptotic protease cascades. Increased integrin ligand binding affinity (activation) is triggered by intracellular signaling events. A Ras-initiated mitogen-activated protein kinase pathway suppresses integrin activation in fibroblasts. We used expression cloning to isolate cDNAs that prevent Ras suppression of integrin activation. Here, we report that PEA-15, a small death effector domain (DED)-containing protein, blocks Ras suppression. PEA-15 does not block the capacity of Ras to activate the ERK mitogen-activated protein kinase pathway. Instead, it inhibits suppression via a pathway blocked by a dominant-negative form of the distinct small GTPase, R-Ras. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to reverse suppression of integrin activation. Thus, certain DED-containing proteins can regulate integrin activation as opposed to apoptotic protease cascades. mitogen-activated protein death effector domain polymerase chain reaction 3′-untranslated region activation index death domain fluorescence-activated cell sorter Chinese hamster ovary hemagglutinin. Integrins are transmembrane heterodimers that mediate cell-cell and cell-extracellular matrix adhesion (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar). The affinity of some integrins for ligand is regulated by "inside-out" cell signaling cascades (2Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 3Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar). Regulation of integrin affinity for ligand (activation) is important in cell migration (4Huttenlocher A. Ginsberg M.H. Horwitz A.F. J. Cell Biol. 1996; 134: 1551-1562Crossref PubMed Scopus (313) Google Scholar), fibronectin matrix assembly (5Wu C. Keivens V.M. O'Toole T.E. McDonald J.A. Ginsberg M.H. Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (299) Google Scholar), platelet aggregation in hemostasis and thrombosis (6Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar), and morphogenesis (7Ramos J.W. Whittaker C.A. DeSimone D.W. Development. 1996; 122: 2873-2883PubMed Google Scholar, 8Martin-Bermudo M.D. Dunin-Borkowski O.M. Brown N.H. J. Cell Biol. 1998; 141: 1073-1081Crossref PubMed Scopus (50) Google Scholar). This cellular regulation of integrin activation is energy-dependent, cell type-specific, and is mediated through integrin cytoplasmic domains (9O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). In fibroblastic cells, activation of the small GTP-binding protein Ha-Ras or its effector kinase, c-Raf-1, initiates a signaling pathway that blocks integrin activation (10Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). This suppressor activity correlates with the activation of the ERK MAP1 kinase pathway and does not require mRNA transcription or protein synthesis. The downstream effectors or regulators of this integrin suppression pathway remain to be identified. Integrin activation is readily measured by the binding of activation-dependent ligands, which can be used as a selective marker in expression cloning schemes. One such scheme (10Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar,11Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (257) Google Scholar) uses a Chinese hamster ovary cell line (αβpy cells) stably expressing a chimeric integrin (αIIbα6Aβ3β1) that contains the extracellular and transmembrane domains of αIIbβ3 fused to the cytoplasmic domains of α6Aβ1. This chimeric integrin has the ligand binding properties of αIIbβ3, and its activation state is regulated through the α6Aβ1 cytoplasmic domains. Consequently, flow cytometry (FACS) can be used to assess the activation state of the chimeric integrin by measuring the binding of fibrinogen or the ligand-mimetic monoclonal antibody, PAC1. To elucidate Ras-induced integrin suppression, we modified this scheme to identify proteins that prevent Ras suppression. Specifically, we used Ras to suppress integrin activation in αβpy cells and isolated co-transfected cDNAs that blocked this suppression. Here we report that PEA-15 (phosphoprotein enriched inastrocytes), a small death effector domain (DED)-containing protein, blocks Ras suppression downstream of MAP kinase via a pathway blocked by a dominant interfering mutant of a distinct small GTPase, R-Ras. αβpy cells are a CHO cell line that expresses the polyoma large T antigen and a constitutively active recombinant chimeric integrin (αIIbα6Aβ3β1) (12Baker E.K. Tozer E.C. Pfaff M. Shattil S.J. Loftus J.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1973-1978Crossref PubMed Scopus (67) Google Scholar). αβpy cells were maintained in Dulbecco's modified Eagle's medium (BioWhitaker, Walkersville, MD) supplemented with 10% fetal calf serum (BioWhitaker), 1% non-essential amino acids (Life Technologies, Inc.), 1% glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 700 μg/ml G418 (Life Technologies, Inc.). The activation-specific anti-αIIbβ3 monoclonal antibody PAC1 (13Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar) was generously provided by Dr. S. Shattil (Scripps Research Institute). The anti-αIIbβ3monoclonal antibody anti-LIBS6 has been described previously (14Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar). The anti-Tac antibody, 7G7B6, was obtained from the American Tissue Culture Collection (Rockville, MD). 7G7B6 was biotinylated with biotin-N-hydroxysuccinimide (Sigma) according to the manufacturer's instructions. The mouse monoclonal anti-HA antibody (12CA5) was produced in our laboratory (15Field J. Nikawa J. Broek D. MacDonald B. Rodgers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (731) Google Scholar). The αIIbβ3–specific peptide inhibitor Ro43-5054 (16Alig L. Edenhofer A. Hadvary P. Hurzeler M. Knopp D. Muller M. Steiner B. Trzeciak A. Weller T. J. Med. Chem. 1992; 35: 4393-4407Crossref PubMed Scopus (202) Google Scholar) was a generous gift from B. Steiner (Hoffmann-La Roche, Basel). The CHO-K1 oligo(dT)-primed library is directionally cloned into pcDNA1 and was obtained from Invitrogen (San Diego, CA). The library is reported to contain 1.8 × 107 primary recombinants. pDCR-Ha-RasG12V was a gift from Dr. M. H. Wigler (Cold Spring Harbor Laboratory). Tac-α5 (17LaFlamme S.E. Thomas L.A. Yamada S.S. Yamada K.M. J. Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (207) Google Scholar) was generously provided by Drs. S. LaFlamme and K. Yamada (National Institutes of Health). HA-Erk2 was described previously (18Renshaw M.W. Lea-Chou E. Wang J.Y.J. Curr. Biol. 1996; 6: 76-83Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Dr. G. Bokoch kindly provided pCMV5-Ha-RasT17N. pcDNA3-R-RasG38V and pcDNA3-R-RasT43N (19Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) were gifts from Dr. E. Ruoslahti (The Burnham Institute, La Jolla, CA) with permission from Dr. A. Hall (University of London). pCHA-MEK2 222/226D was provided by Dr. M. Weber (University of Virginia). Expression cloning was done using αβpy cells. αβpy cells were divided into 18 subconfluent 100-mm plates and co-transfected with Tac-α5 (2 μg/plate), Ha-RasG12V (3 μg/plate) and a CHO-K1 library (4 μg/plate) using LipofectAmineTM (Life Technologies, Inc.). 48 h after transfection, cells were collected and stained for FACs analysis with antibodies PAC1 and 7G7B6 as described previously (9O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). Cells that bound high levels of both PAC1 and 7G7B6 were collected by fluorescence-activated cell sorting (FACSTAR, Becton Dickinson). Plasmid DNA was extracted from collected cells by Hirt Supernatant (20Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3348) Google Scholar). This plasmid DNA was used to transform Escherichia coli MC1061/P3 cells. Bacterial colonies were grown, stored, and pooled into groups of 16 for plasmid purification (Qiagen, Chatsworth, CA) and analysis. To isolate single cDNAs that reverse Ras suppression of PAC1 binding, groups of cDNAs were transfected into αβpy cells along with Ha-RasG12V and Tac-α5. Transfectants were screened by two-color flow cytometry (FACScalibur, Becton Dickinson) as described above. A group containing cDNAs that reverse Ras suppression was identified and divided into groups of four for further screening. Positive groups were finally screened as single cDNAs. An HA-tagged PEA-15 lacking the 3′-UTR was created by PCR of CHO PEA-15-pcDNA1 clone using pfu polymerase (New England Biolabs). The amplified product was subcloned into the BamHI/EcoRI sites of pcDNA3. HA-tagged DED and C-terminal domains of PEA-15 were similarly constructed by PCR. PEA-15(D74A) was constructed using the Quickchange kit (Stratagene). DD-PEA-15 was constructed by splice-overlap PCR with pcDNA3-FADD and CHO PEA-15-pcDNA1 as templates. The insert was subcloned into the BamHI/EcoRI sites of pcDNA3. Mutations were verified by sequencing. Analytical two-color flow cytometry was done as described (9O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). In transiently transfected αβpy cells, PAC1 binding was determined for transfected cells (cells positive for the co-transfected Tac-α5 as measured by 7G7B6 binding). Integrin activation was quantitated as an activation index (AI) defined as 100 × (F −F r)/(F LIBS6 −F r) in which F is the median fluorescence intensity of PAC1 binding; F r is the median fluorescence intensity of PAC1 binding in the presence of competitive inhibitor (Ro43-5054, 1 μm); andF LIBS6 is the median fluorescence intensity in the presence of anti-LIBS6 (2 μm). From this we calculated the percent inhibition as 100 × (AI − AIS)/AI, in which AI is the activation index of control cells and AIS is the activation index in the presence of a transfected suppressing cDNA. For ERK kinase assays, αβpy cells were transfected with HA-ERK2 (2 μg) along with test cDNA such as pcDNA3-PEA15 (3 μg) using LipofectAmineTM (20 μl/plate, Life Technologies, Inc.). In instances where more than one test plasmid is used, the amount of DNA transfected was standardized by addition of pcDNA1 control vector. Cells were lysed 48 h after transfection in ice-cold M2 buffer (0.5% Nonidet P-40, 20 mm Tris, pH 7.6, 250 mm NaCl, 5 mmEDTA, 3 mm EGTA, 20 mm sodium phosphate, 20 mm sodium pyrophosphate, 3 mmβ-glycerophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, and 10 μg/ml each of leupeptin and aprotinin). ERK2 activity was measured by an immune-complex assay (from 100 μg of cell lysate protein) using myelin basic protein as a substrate (18Renshaw M.W. Lea-Chou E. Wang J.Y.J. Curr. Biol. 1996; 6: 76-83Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). ERK2 activity was determined by autoradiography. To elucidate the mechanism of Ras-mediated suppression of integrin activation, we used an expression cloning strategy to identify proteins that prevent Ras suppression (Fig.1 A). αβpy cells were co-transfected with activated Ha-Ras and a CHO cell cDNA library. We used flow cytometry to isolate cells that still bound the activation-specific anti-αIIbβ3 antibody, PAC-1, despite transfection with activated Ha-Ras (Fig. 1 B,left panel, box). Seventy-nine cDNAs were recovered from isolated cells. One of these cDNAs, R36, restored PAC1 binding in cells transfected with activated Ras (Fig. 1 B, middle panel). Indeed, R36 transfection resulted in FACS profiles similar to those observed in the absence of Ras suppression (Fig. 1 B, right panel). Ras expression levels in the PEA-15 transfected cells remained comparable to cells transfected with Ras alone (Fig.1 C). Thus, the reversal of Ras suppression was not due to a loss of Ras expression. R36 contained 1,700 base pairs encoding an open reading frame of 130 amino acids (GenBankTM accession number AF080001). A BLAST data base search indicated that the 130-amino acid sequence is 99% identical to mouse phosphoprotein enriched in astrocytes (PEA-15, Fig.2 A) (21Danziger N. Yokoyama M. Jay T. Cordier J. Glowinski J. Chneiweiss H. J. Neurochem. 1995; 64: 1016-1025Crossref PubMed Scopus (95) Google Scholar). The first 80 amino acids of PEA-15 correspond to the canonical DED sequence found in proteins that regulate apoptotic signaling pathways (22Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2158) Google Scholar, 23Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 24Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar) (Fig. 2,A and B). In fact, the PEA-15 DED is more similar to that of FADD than that of the viral DED-containing protein MC159 (Fig. 2 B). The remaining 50 amino acids contain a serine (Ser-104) that is phosphorylated by protein kinase C (25Araujo H. Danziger N. Cordier J. Glowinski J. Chneiweiss H. J. Biol. Chem. 1993; 268: 5911-5920Abstract Full Text PDF PubMed Google Scholar) and a serine (Ser-116) phosphorylated by calcium calmodulin kinase II (26Kubes M. Cordier J. Glowinski J. Girault J.A. Chneiweiss H. J. Neurochem. 1998; 71: 1307-1314Crossref PubMed Scopus (76) Google Scholar). No function has yet been ascribed to PEA-15. The sequence of R36 also contained a predicted 1190-base pair 3′-UTR containing a polyadenylation signal and poly(A) tract. The final 1050 bases of this region are 70% identical to MAT1, a transforming cDNA isolated from a lithium-induced mouse mammary tumor (27Bera T.K. Guzman R.C. Miyamoto S. Panda D.K. Sasaki M. Hanyu K. Enami J. Nandi S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9789-9793Crossref PubMed Scopus (22) Google Scholar). To determine whether the 3′-UTR is necessary for the reversal of Ha-Ras integrin suppression, we tested a construct without this sequence and found that it functioned like the full-length cDNA (data not shown). More than half of the PEA-15 protein consists of a conserved DED (Fig. 2,A and B). This domain, to date, is associated with proteins involved in apoptosis (28Goltsev Y.V. Kovalenko A.V. Arnold E. Varfolomeev E.E. Brodianskii V.M. Wallach D. J. Biol. Chem. 1997; 272: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 29Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 30Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2223) Google Scholar, 31Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4553) Google Scholar). To determine if the DED of PEA-15 is necessary or sufficient for PEA-15 reversal of Ras suppression, we overexpressed mutant forms of PEA-15 in αβpy cells in this assay. Overexpression of only the DED of PEA-15 did not reverse Ras suppression (Fig. 2 C). It is therefore not sufficient for this function. Mutants of PEA-15 lacking the DED (C-Term) were also unable to reverse Ras suppression (Fig. 2 C). Furthermore, a conserved aspartate is present in a RxDLL sequence in all DEDs (32Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2109) Google Scholar) (asterisk in Fig. 2 B). Mutation of this aspartate (D74A) prevented PEA-15 reversal of Ras suppression (Fig.2 C). The structure of the DED is similar to that of the death domain (DD) of FADD (33Eberstadt M. Huang B. Chen Z. Meadows R.P. Ng S.C. Zheng L. Lenardo M.J. Fesik S.W. Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar). Substitution of the DD of FADD for the DED of PEA-15 yielded a chimeric molecule incapable of reversing Ras suppression (Fig. 2 C). In all cases, mutant PEA-15 constructs were expressed (Fig. 2 D). The DED of PEA-15 is therefore necessary, but not sufficient, for reversal of Ras suppression. Additionally, substitution of the DED of PEA-15 with the DED of FADD resulted in a chimeric protein that induced apoptosis (data not shown). This indicates that the DED of PEA-15 is functionally distinct from that of FADD and contains primary sequence information required for PEA-15 function. Thus, our studies define a new function for DEDs. Ras suppresses integrin activation by activating a MAP kinase pathway (10Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). When activated Ras (RasG12V) and PEA-15 were co-expressed, the integrins were not suppressed although the activated variant of Ras was present. Consequently, we assessed the effect of PEA-15 on other activated components of the MAP kinase pathway. Exchange factor mediated GTP loading, and activation of Ras initiates the MAP kinase pathway (34Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar,35Robinson M.J. Cheng M. Khokhlatchev A. Ebert D. Ahn N. Guan K.L. Stein B. Goldsmith E. Cobb M.H. J. Biol. Chem. 1996; 271: 29734-29739Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Active Ras recruits and thus activates Raf kinase. We found that PEA-15 could reverse suppression initiated by an activated Raf (RafCAAX, Fig. 3 A), indicating that its site of action is distal to Raf activation. Raf phosphorylates and activates MEK, which in turn activates ERK. PEA-15 also rescued suppression mediated by activated MEK (MEK2 222/226D, Fig.3 A) and did not block ERK activation (Fig. 3 B). This suggests that PEA-15 function is distal to ERK. PEA-15 might reverse Ras suppression by activating effectors that oppose Ras signaling to integrins. R-Ras is a Ras-related GTP-binding protein that activates integrins (19Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) and reverses Ha-Ras suppression of integrin activation. 2T. Sethi, M. Ginsberg, J. Downward, and P. Hughes, submitted for publication. Like PEA-15, R-Ras does not affect Ha-Ras activation of Erk. Consequently, we asked if the capacity of PEA-15 to reverse suppression depends on R-Ras. A dominant-negative R-Ras (R-RasT43N) blocked the ability of PEA-15 to reverse suppression by activated Ha-Ras (Fig.4 A). Cells expressing the dominant-negative R-RasT43N had moderately reduced expression of PEA-15 and Ha-Ras (Fig. 4 B); however Ha-Ras expression levels remained sufficient to suppress integrin activity (Fig. 4 A). Thus, the reduced expression levels of Ha-Ras do not account for the effects of dominant-negative R-Ras. Furthermore, activated R-Ras still reversed suppression when co-expressed with the dominant-negative R-RasT43N (Fig. 4 A). Therefore, the effect of R-RasT43N was upstream of R-Ras. These results indicate that PEA-15 inhibition of Ha-Ras suppression is impaired by expression of a dominant-negative R-Ras construct. Because R-Ras and Ha-Ras are similar (36Lowe D.G. Capon D.J. Delwart E. Sakaguchi A.Y. Naylor S.L. Goeddel D.V. Cell. 1987; 48: 137-146Abstract Full Text PDF PubMed Scopus (157) Google Scholar), they may share some of the same guanine-nucleotide exchange factors (37Gotoh T. Niino Y. Tokuda M. Hatase O. Nakamura S. Matsuda M. Hattori S. J. Biol. Chem. 1997; 272: 18602-18607Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), but R-Ras is also regulated by effectors and activators distinct from those that control Ha-Ras (38Huff S.Y. Quilliam L.A. Cox A.D. Der C.J. Oncogene. 1997; 14: 133-143Crossref PubMed Scopus (43) Google Scholar). The dominant-negative form of R-Ras we used probably sequesters GEFs (39Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1756) Google Scholar). Consistent with this site of action, DN-R-Ras failed to affect rescue mediated by an activated variant of R-Ras (G38V). Furthermore, a DN-Ha-Ras construct did not affect PEA-15 reversal of suppression. Hence, the DN-R-Ras acts by blocking events specific for R-Ras and not ones common to both Ha-Ras and R-Ras. Similarly, dominant-negative constructs of the small GTP-binding proteins Cdc42, Rac, and Rho did not prevent PEA-15 reversal of Ras suppression (data not shown), further suggesting that the effect is R-Ras-specific. R-Ras activates integrins (19Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) and reverses Ha-Ras suppression of integrin activation (Fig. 4 A). Therefore, PEA-15 may reverse Ras suppression via an R-Ras-dependent mechanism. The proteins involved in R-Ras regulation remain unclear (40Bos J.L. Biochim. Biophys. Acta. 1997; 1333: M19-M31Crossref PubMed Scopus (136) Google Scholar). However, our data suggest that PEA-15 could be a novel upstream regulator of R-Ras activity. Alternatively, the effects of the dominant-negative R-Ras construct may be due to interference with the closely related protein TC21/R-Ras2 (41Graham S.M. Cox A.D. Drivas G. Rush M.G. D'Eustachio P. Der C.J. Mol. Cell. Biol. 1994; 14: 4108-4115Crossref PubMed Scopus (92) Google Scholar, 42Chan A.M. Miki T. Meyers K.A. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7558-7562Crossref PubMed Scopus (86) Google Scholar). It will be interesting to analyze the potential interplay between PEA-15 and R-Ras. In summary, we used an expression cloning scheme to identify proteins that prevent Ha-Ras suppression of integrin activation. We report that the DED-containing protein, PEA-15, blocks Ras suppression. PEA-15 does not inhibit Ras activation of the ERK MAP kinase pathway, but rather blocks Ras suppression via a pathway inhibited by a dominant-negative form of R-Ras. Finally, the DED of PEA-15 is necessary, but not sufficient, for the reversal of Ras suppression. Hence, these data provide evidence that DED-containing proteins can regulate integrin activation as well as apoptosis. Moreover, we have identified PEA-15 as a novel regulator of inside-out integrin signaling pathways. We thank our colleagues for their generosity in providing the reagents listed under "Experimental Procedures." We are also grateful to Dr. Sandy Shattil and Dr. Martin Schwartz for their critical review of the manuscript.
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