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hSos1 Contains a New Amino-terminal Regulatory Motif with Specific Binding Affinity for Its Pleckstrin Homology Domain

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m204423200

ISSN

1083-351X

Autores

Rocı́o Jorge, Natasha Zarich, José Luís Oliva, Marta Azañedo, Natalia J. Martinez, Xavier de la Cruz, José M. Rojas,

Tópico(s)

Biochemical and Molecular Research

Resumo

The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associatedin vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain. The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associatedin vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain. Withdrawal: hSos1 contains a new amino-terminal regulatory motif with specific binding affinity for its pleckstrin homology domain.Journal of Biological ChemistryVol. 293Issue 29PreviewVOLUME 277 (2002) PAGES 44171–44179 Full-Text PDF Open Access Sos guanine nucleotide exchange proteins mediate Ras activation induced by various tyrosine kinase receptors (1Boguski M. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar). Sos consists of several defined domains, each of which has a distinct function. For example, nucleotide exchange activity on Ras is mediated by a central domain of Sos that, among the various Ras guanine nucleotide exchange factors, is very well conserved (1Boguski M. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar) and for which the structure has been determined in complex with Ras (2Boriack-Sjodin P.A. Margarit S.M. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (637) Google Scholar). The carboxyl-terminal region of Sos is proline-rich and contains specific sequences (PΨΨPPR) that bind the SH3 (Src homology 3) domains of Grb2 (3Chardin P. Camonis J.H. Gale N.W. van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (674) Google Scholar, 4Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (818) Google Scholar). Finally, the amino-terminal region of Sos is ∼600 amino acids long and contains regions of homology to Dbl (DH) 1The abbreviations used are: DH, Dbl homology; PH, pleckstrin homology; HF, histone fold; GEF, guanine exchange factor; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; wt, wild type; NDP, region comprising the N terminus + DH + PH; PKD, protein kinase D; RBD, ras binding domain; GRF, guanine releasing factor and pleckstrin (PH) domains involved in Rac1 activation (5Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (390) Google Scholar, 6Scita G. Nordstrom J. Carbone R. Tenca P. Giardina G. Gutkind S. Bjarnegard M. Betsholtz C. Di Fiore P.P. Nature. 1999; 401: 290-293Crossref PubMed Scopus (286) Google Scholar) and phospholipid binding (7Chen R.H. Corbalan-Garcia S. Bar-Sagi D. EMBO J. 1997; 16: 1351-1359Crossref PubMed Scopus (116) Google Scholar), respectively, for which the structures have also been determined (8Koshiba S. Kigawa T. Kim J.H. Shirouzu M. Bowtell D. Yokoyama S. J. Mol. Biol. 1997; 269: 579-591Crossref PubMed Scopus (47) Google Scholar, 9Soisson S.M. Nimnual A.S., Uy, M. Bar-Sagi D. Kuriyan J. Cell. 1998; 95: 259-268Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Stimulation of cells with growth factor leads to the association of Sos-Grb2 complexes with activated receptors, leading to the stimulation of Ras through the juxtaposition of Sos and Ras at the membrane (1Boguski M. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar). Sos-Ras binding involves the switch 1 and switch 2 regions of Ras (2Boriack-Sjodin P.A. Margarit S.M. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (637) Google Scholar). Whereas the interaction with switch 2 mediates the anchoring of Ras to Sos, the interaction with switch 1 leads to the disruption of the nucleotide-binding site and GDP dissociation (10Hall B.E. Yang S.S. Boriack-Sjodin P.A. Kuriyan J. Bar-Sagi D. J. Biol. Chem. 2001; 276: 27629-27637Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In this model, both the cytosolic and membrane-bound Sos forms are thought to exhibit similar nucleotide exchange activity; no variation of this activity is thought to occur as a consequence of relocation inside the cell. In support of this idea, constitutive or conditional membrane targeting of these exchange factors has been shown to strengthen Ras activation in transfected cells (11Aronheim A. Engelberg L., Al- Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (431) Google Scholar, 12Quilliam L.A. Huff S.Y. Rabun K.M. Wei W. Park W. Broek D. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8512-8516Crossref PubMed Scopus (129) Google Scholar). However, some reports suggest that regardless of sub-cellular location, the intrinsic Ras guanine nucleotide exchange activity of Sos (Ras-GEF activity) may differ before and after stimulation of surface tyrosine kinase receptors (13Li B. Subleski M. Fusaki N. Yamamoto T. Copeland T. Princler G.L. Kung H. Kamata T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1001-1005Crossref PubMed Scopus (43) Google Scholar, 14Rojas J.M. Subleski M. Coque J.J. Guerrero C. Saez R., Li, B.Q. Lopez E. Zarich N. Aroca P. Kamata T. Santos E. Oncogene. 1999; 18: 1651-1661Crossref PubMed Scopus (12) Google Scholar). Several reports suggest that the carboxyl-terminal portion of Sos exerts negative regulation over the activity of Sos1 (15Corbalan-Garcia S. Margarit S.M. Galron D. Yang S.S. Bar-Sagi D. Mol. Cell. Biol. 1998; 18: 880-886Crossref PubMed Scopus (85) Google Scholar, 16McCollam L. Bonfini L. Karlovich C.A. Conway B.R. Kozma L.M. Banerjee U. Czech M.P. J. Biol. Chem. 1995; 270: 15954-15957Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Byrne J.L. Paterson H.F. Marshall C.J. Oncogene. 1996; 13: 2055-2065PubMed Google Scholar, 18Karlovich C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (77) Google Scholar, 19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar). However, the role of the amino-terminal region of Sos is not fully understood. Thus, some reports suggest that this region is responsible for positive regulation of Sos1 activity (17Byrne J.L. Paterson H.F. Marshall C.J. Oncogene. 1996; 13: 2055-2065PubMed Google Scholar, 18Karlovich C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (77) Google Scholar), basically through the cytoplasmic membrane localization of Sos1 protein. Nevertheless, stable membrane association of Sos by addition of a myristoylation signal to this protein, still lacking the amino-terminal region, is not sufficient for Sos to be biologically active (20Qian X. Vass W.C. Papageorge A.G. Anborgh P.H. Lowy D.R. Mol. Cell. Biol. 1998; 18: 771-778Crossref PubMed Scopus (54) Google Scholar). Others reports suggest that the amino-terminal portion of Sos1 is involved in the negative regulation of its catalytic activity and exerts negative allosteric control on the interaction of the Sos catalytic domain with Ras (15Corbalan-Garcia S. Margarit S.M. Galron D. Yang S.S. Bar-Sagi D. Mol. Cell. Biol. 1998; 18: 880-886Crossref PubMed Scopus (85) Google Scholar). Most studies of the amino-terminal region of Sos1 focus on the DH and PH domains and disregard other possibilities such as the peptide region upstream of DH. The aim of the present study is to explore the function of the amino-terminal region of hSos1, specifically the small region (200 residues) upstream of DH domain, as well as to examine further its involvement in the regulation of hSos1 activity. Toward these ends, we characterized the effect of this region on biological and biochemical signaling events related to Ras activation. In addition, we analyzed its in vitro and in vivo specific association with the PH domain of hSos1 and its function in the physiological activity of hSos1. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% calf serum (Invitrogen). COS1 and the human 293T (kidney keratinocyte) cell lines were maintained in DMEM supplemented with 10% fetal calf serum (fetal calf serum, Invitrogen). Transient transfections in COS1 and 293T cells were performed with the LipofectAMINE reagent (Invitrogen). COS1 cells for serum starvation received DMEM containing 0.5% fetal bovine serum 24 h after transfection and were then incubated for another 24 h. All assays were done 48 h after transfection. NIH3T3 cells were transfected (transient or stable) by the calcium phosphate precipitation technique. Morphologically transformed foci were scored after 2–3 weeks in culture (21Rojas J.M. Coque J.J.R. Guerrero C. Aroca P. Font de Mora J. de la Cruz X. Lorenzi M.V. Esteban L.M. Santos E. Oncogene. 1996; 12: 2291-2300PubMed Google Scholar). Transfected cells were also selected in medium supplemented, as appropriate, with Geneticin 750 μg/ml (Invitrogen). Monoclonal antibody to phospho-MAPK protein was purchased from New England Biolabs. Rabbit polyclonal antiserum to MAPK (ERK1/ERK2) and rabbit anti-GST polyclonal antibody came from Santa Cruz Biotechnology Inc., monoclonal anti-His, from Sigma, and anti-HA and anti-AU5 monoclonal antibodies, from the Berkeley Antibody Co. The plasmids pCEFL-KZ-HA, pCEFL-KZ-AU5, pCEFL-KZ-HA-hSos1, pCEFL-KZ-HA-NDP, pCEFL-KZ-AU5-H-Ras wt, pCEFL-KZ-AU5-H-RasV12, pGEX-4T-1-NDP, pGal4-Luc, and pCDNAIII-Gal4-Elk-1 were described previously (14Rojas J.M. Subleski M. Coque J.J. Guerrero C. Saez R., Li, B.Q. Lopez E. Zarich N. Aroca P. Kamata T. Santos E. Oncogene. 1999; 18: 1651-1661Crossref PubMed Scopus (12) Google Scholar, 19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar, 21Rojas J.M. Coque J.J.R. Guerrero C. Aroca P. Font de Mora J. de la Cruz X. Lorenzi M.V. Esteban L.M. Santos E. Oncogene. 1996; 12: 2291-2300PubMed Google Scholar). The HF motif, DH, PH, and DH-PH domains of hSos1 (coding regions 1–640, 537–1326, 1263–1701, and 537–1653, respectively) were PCR-amplified from pCEFL-KZ-HA-hSos1 using the specific primers and providing sitesBglII and NotI at the 5′ and 3′ ends, respectively. The amplified products were then subcloned intoBglII and NotI sites of vectors pCEFL-KZ-HA and pCEFL-KZ-AU5 and into BamHI and NotI sites in pGEX-4T-1 (Amersham Biosciences). Likewise, the HF motif was PCR-amplified with specific primers providing sites BamHI and SalI at the 5′ and 3′ ends, respectively, and subcloned into BamHI and SalI sites of pQE30 vector (Qiagen). The myristoylated construct Myr-HA-NDP was obtained from pCEFL-KZ-HA-NDP by digestion with BamHI andNotI, and the small fragmentBamHI-NotI (HA-NDP) was then subcloned between sites BamHI and NotI of plasmid pCEFL-KZ-Myr (kindly provided by J. S. Gutkind, NIDCR, National Institutes of Health) containing the myristoylation signal of Src. The NDP truncated mutant of hSos1 (ΔNDP-hSos1) was generated using primers for positions 1702–4000 of hSos1 and providing restriction sitesBclI and NotI at the 5′ and 3′ ends and subcloned into sites BglII and NotI of vector pCEFL-KZ-HA. To obtain the HF truncated mutant of hSos1 (ΔHF-hSos1), the plasmid pCEFL-KZ-HA-DH-PH was digested with the restriction endonucleasesHindIII and SpeI, and the HA-DH-PH fragment was subcloned into the HindIII and SpeI sites of pCEFL-KZ-HA-hSos1. K-Ras wt, K-RasV12, N-Ras wt, and N-RasV12 were PCR-amplified from pCDNA-K-Ras wt, pCDNA-K-RasV12, pCDNA-N-Ras wt, and pCDNA-N-RasV12, respectively (kind gift of J. S. Gutkind), using the specific primers providing sitesBglII and NotI at the 5′ and 3′ ends. The amplified products were then subcloned into BglII andNotI sites of vector pCEFL-KZ-AU5. The sequences of the oligonucleotides utilized are available upon request. S. J. Taylor (Cornell University) kindly provided the plasmid pGEX-RBD containing the Raf-Ras-binding domain (amino acids 1–149) fused to glutathione S-transferase (GST). The GST-RBD protein was purified (from Escherichia coli BL21(DE3) harboring that plasmid) following the method described previously (19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar). Similarly, the BL21(DE3) strain of E. coli was transformed with the vector pGEX-4T-1 encoding the fusion protein GST-NDP, GST-HF, GST-DH, GST-PH, GST-DH-PH (all containing domains of hSos1), or GST-PH2 (containing the PH domain 2 of Ras-GRF1) kindly provided by J. S. Gutkind. This bacterial strain was also transformed with the vector pGEX-4T-3 encoding the fusion protein GST-PH (containing the PH domain of PKD) kindly provided by T. Iglesias (22Waldron R.T. Iglesias T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Protein purification was performed according to the method described previously (21Rojas J.M. Coque J.J.R. Guerrero C. Aroca P. Font de Mora J. de la Cruz X. Lorenzi M.V. Esteban L.M. Santos E. Oncogene. 1996; 12: 2291-2300PubMed Google Scholar). The M15 strain ofE. coli harboring plasmid pREP4 (Qiagen) was transformed with the vector pQE30 encoding the HF motif of hSos1 containing six consecutive histidine residues (the 6xHis tag) at their amino terminus. The 6xHis-HF peptide was purified as described previously (23Font de Mora J. Guerrero C. Mahadevan D. Coque J. Rojas J. Esteban L. Rebecchi M. Santos E. J. Biol. Chem. 1996; 271: 18272-18276Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Fifty pmol of 6xHis-HF protein, purified and eluted, were incubated with 50 pmol of either GST-NDP, GST-HF, GST-DH, GST-PH, GST-DH-PH of hSos1, GST-PH2 of Ras-GRF1, GST-PH of PKD, or GST proteins coupled to glutathione-Sepharose beads and incubated for 60 min at 4 °C following the methodology described previously (21Rojas J.M. Coque J.J.R. Guerrero C. Aroca P. Font de Mora J. de la Cruz X. Lorenzi M.V. Esteban L.M. Santos E. Oncogene. 1996; 12: 2291-2300PubMed Google Scholar). Transfected cells, some stimulated for 10 min with 30% fetal calf serum, were lysed in cold lysis buffer containing 25 mm HEPES, pH 7.5, 1% Triton X-100, 150 mm NaCl, 10 mmMgCl2, 1 mm sodium orthovanadate (Na3VO4), 25 mm NaF, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin, aprotinin, pepstatin A, and trypsin inhibitor. Nucleus-free supernatants were incubated with GST-RBD on glutathione-Sepharose beads and analyzed as previously described (19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar). NIH3T3 cells were transfected with 0.6 μg of constructs encoding for either hSos1 or Ras, together with 16 ng of pCDNAIII-Gal4-Elk-1, 0.1 μg of pRL-TK (a plasmid expressing the enzyme Renilla luciferase), and 0.3 μg of the reporter plasmid (pGal4-Luc). Cells were kept for 24 h in DMEM supplemented with 0.5% calf serum and 18 h later were stimulated for 8 h with 30% fetal calf serum. The assays were performed as described previously (19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar). To ascertain the function of the amino-terminal half of hSos1, we cloned this region (NDP, residues 1–600; NDP = Nter + DH + PH) in a mammalian expression vector and also generated a truncated mutant of this region (ΔNDP-hSos1). The NDP region includes the DH and PH domains and a small amino-terminal (Nter) region (residues 1–200) upstream of DH. This amino-terminal region displays very close sequence similarity to histone H2A (24Baxevanis A.D. Arents G. Moudrianakis E.N. Landsman D. Nucleic Acids Res. 1995; 23: 2685-2691Crossref PubMed Scopus (182) Google Scholar) (the core of homology are residues 90–173), denoted by us as HF (histone fold) motif (Fig.1). A classical effect of the Ras pathway activation is to elicit formation of transformed foci in NIH3T3 cells. We cotransfected NIH3T3 cells with the hSos1 constructs and H-Ras wt as described above and assessed the transformed foci (Fig. 1). The difference in transforming activity between hSos1 wt and the NDP truncated mutant was shown in experiments in which the hSos1 constructs were cotransfected with normal ras genes. The over-expression of H-Ras wt alone produced weak but reproducible transforming activity, which was enhanced severalfold when hSos1 wt was included in the cotransfection experiments (Fig. 1). However, over-expression of ΔNDP-hSos1 (Fig. 1 A) or NDP region (Fig. 1 B) together with H-Ras wt consistently resulted in a significant reduction in the number of transformed foci produced by normal H-Ras alone. These results suggest that hSos1 needs its amino-terminal half (NDP region) for the synergistic effect with H-Ras wt. This NDP region was involved in the positive control of hSos1 function, in agreement with previously published observations on the regulatory effect of the amino-terminal region of Sos (17Byrne J.L. Paterson H.F. Marshall C.J. Oncogene. 1996; 13: 2055-2065PubMed Google Scholar, 18Karlovich C.A. Bonfini L. McCollam L. Rogge R.D. Daga A. Czech M.P. Banerjee U. Science. 1995; 268: 576-579Crossref PubMed Scopus (77) Google Scholar, 20Qian X. Vass W.C. Papageorge A.G. Anborgh P.H. Lowy D.R. Mol. Cell. Biol. 1998; 18: 771-778Crossref PubMed Scopus (54) Google Scholar). To determine whether the ability to reduce the number of transformed foci induced by normal H-Ras can be assigned to any particular region of NDP, we over-expressed the different domains (HF, DH-PH, DH, and PH) together with H-Ras wt. Thus, ectopic expression of HF, DH-PH, and DH domains led to the same effect as the complete NDP region, inhibiting the transforming activity of H-Ras wt (Fig. 1 B). In sharp contrast, overexpression of the PH domain had no effect on the number of transformed foci (Fig. 1 B). Further, cotransfection of the truncated mutant ΔHF-hSos1 (hSos1 without HF motif) together with H-Ras wt more actively induced focus formation than hSos1 wt (Fig. 1 A). The ectopic expression of the different hSos1 constructs did not affect the expression levels of endogenous Sos1 (data not shown). Taken together, these results suggest that the HF motif of hSos1 protein is involved in the negative control of hSos1 activity. We confirmed the negative effect of the HF motif on Sos activity by evaluating its function in the signaling pathways downstream of Sos. Specifically, we investigated whether over-expression of the NDP region or its different domains (HF, DH-PH, DH, and PH) affected the Raf-MEK-MAPK pathway. Therefore, 293T cells were transiently transfected with full-length hSos1 (hSos1 wt), the truncated mutants ΔHF-hSos1 and ΔNDP-hSos1, the NDP region, or their different domains. After serum starvation, cells were stimulated with epidermal growth factor (EGF, Fig.2 A). As a positive control we used the oncogenic version of H-Ras (H-RasV12). The results show that over-expression of full-length hSos1 induces, under starved conditions, a level of ERK activation comparable with that seen for naive cells stimulated by epidermal growth factor, whereas ΔHF-hSos1 appears more potent than full-length hSos1 in eliciting this response. However, the over-expression of ΔNDP-hSos1, NDP region, HF, DH-PH and DH domains, but not the PH domain, led to a significant reduction of activated ERK1/ERK2 (Fig. 2 A). This inhibitory effect on the MAP kinase pathway produced by over-expression of NDP occurs upstream of Ras because the cotransfection of H-RasV12 and NDP induced similar levels of activated ERK1/ERK2 as H-RasV12 alone (Fig. 2 A). The same results were obtained with ΔNDP-hSos1 mutant and by the HF, DH-PH, or DH domains (data not shown). To confirm the results presented above, the NDP region and its domains were studied for their ability to affect the MAPK pathway. We used a reporter assay in NIH3T3 cells cotransfected with hSos1 or H-RasV12 constructs, together with a chimerical Gal4-Elk1 transcription factor and the reporter plasmid TATA-Gal4-Luc. Fig. 2 B shows the results obtained in a set of experiments in which we measured the induction of luciferase activity under starved and serum-stimulated conditions. In accordance with the above p44/p42 MAP kinase results, we also detected inhibition of serum-induced Gal4-Elk1 activation by the NDP region of hSos1 or its domains, excepting the PH domain. In the case of NDP, the inhibitory effect was also detected with a version constitutively targeted to the cell membrane (NDP-myristoylated) (Fig.2 B). Finally, according to the phospho-ERK detection assays, over-expression of the NDP truncated mutant of hSos1 (ΔNDP-hSos1) strongly inhibited serum-induced Gal4-Elk1 activation (Fig.2 C), whereas the truncated mutant ΔHF-hSos1 was more potent than hSos1 wt in inducing Gal4-Elk1 activation (Fig.2 C). Because the inhibitory effect on the MAP kinase activation produced by the over-expression of NDP occurs upstream of Ras, we investigated whether the overexpression of the NDP region of hSos1 (or its domains) affects Ras activation induced by mitogenic stimulation. As a read-out, we measured the level of Ras-GTP in transient cotransfectants overexpressing hSos1 or Ras wt. After transient transfection, COS1 cells were serum-starved for 18 h and then stimulated with fetal calf serum (Fig.3). The Ras-GTP levels were detected using a nonradioactive Ras-GTP detection assay (19Zarich N. Oliva J.L. Jorge R. Santos E. Rojas J.M. Oncogene. 2000; 19: 5872-5883Crossref PubMed Scopus (19) Google Scholar). Fig. 3 Ashows representative results wherein we compared the levels of Ras-GTP in transient cotransfected COS1 cells expressing AU5-K-Ras wt and HA-tagged constructs of either full-length hSos1 or the NDP region (or their domains), under both basal and mitogenic conditions. As controls, COS1 cells harboring AU5-K-Ras wt alone or AU5-K-RasV12 were analyzed in parallel. As expected, analysis of GTP-bound Ras in cell lysates expressing full-length hSos1 showed Ras activation under basal conditions (Fig. 3 A). However, the over-expression of the NDP region of hSos1 or any of its domains, with the exception of the PH domain, inhibited AU5-K-Ras wt activation induced by serum stimulation (Fig. 3 A). Again, this inhibition was also observed with a version of NDP constitutively targeted to the cell membrane (NDP-myristoylated) (Fig. 3, A and B). This assay was performed with the three types of mammalian Ras (K-, H-, and N-Ras). Fig. 3 B summarizes the results obtained when we analyzed the AU5-Ras-GTP/AU5-Ras levels under basal and stimulated conditions. In line with the focus formation and MAP kinase results, whereas Ras activation was completely blocked by the over-expression of NDP and HF peptides, the levels of Ras-GTP upon serum stimulation were unaffected by ectopic expression of PH domain (Fig. 3 B). Nevertheless, the inhibitory effect on Ras activation due to the over-expression of DH-PH and DH domains was more clearly detected with K-Ras than with H-Ras or N-Ras (Fig. 3 B). Given that the truncated mutant ΔHF-hSos1 was more potent than the full-length hSos1 inducting transforming foci (Fig. 1) and MAP kinase activation (Fig. 2), we decided to determine whether this mutant was also more efficient in inducing Ras activation. To this end, COS1 cells were cotransfected with AU5-H-Ras wt and either full-length HA-hSos1 or HA-ΔHF-hSos1, and Ras-GTP levels were detected as described above. As controls, COS1 cells transfected with AU5-H-Ras wt alone or AU5-H-RasV12 were analyzed in parallel. Analysis of GTP-bound Ras in cell lysates expressing either full-length hSos1 or ΔHF-hSos1 showed Ras activation under basal conditions (Fig.4 A). The amounts of AU5-H-Ras-GTP under basal and stimulated conditions, standardized to AU5-H-Ras levels (Fig. 4 B), in the cells containing HA-hSos1 wt were higher than in cells transfected with AU5-H-Ras wt alone but lower than in cells containing AU5-H-RasV12. Interestingly, under the same basal and stimulated conditions, HA-ΔHF-hSos1 induced Ras-GTP levels to a greater extent than full-length hSos1 (Fig. 4 B), which is in agreement with the results observed in focus formation and MAP kinase activation assays. Taken together, the above results suggest that the HF motif of hSos1 exerts a negative regulatory effect on hSos1 activity. Because the NDP region (containing HF, DH, and PH domains) is necessary for hSos1 function, a possible mechanism to explain this negative effect could be through molecular interactions of the HF motif with the domains of the NDP region. To test this hypothesis, we analyzed the in vitro interaction between HF and NDP as well as with each one of its corresponding domains. The NDP region or its domains (HF, DH, DH-PH, PH) were expressed as GST fusion proteins, and the HF motif was also expressed as a 6xHis fusion peptide. The purified 6xHis-HF peptide was incubated with similar amounts of purified GST, GST-HF, GST-DH, GST-DH-PH, GST-PH, and GST-NDP proteins coupled to glutathione-Sepharose beads, and the proteins bound to the beads were analyzed by immunoblotting with antibodies to 6xHis (Fig.5 A). Whereas purified GST alone, GST-HF, and GST-DH (Fig. 5 A) did not bind any HF, high amounts of 6xHis-HF bound to GST-PH beads (Fig. 5 A). Consistent with these results, GST-DH-PH and GST-NDP proteins (both containing PH domain) also bound HF (Fig. 5 A). The yeast two-hybrid approach (data not shown) gave the same results. The specificity of the in vitro interaction between the HF motif and PH domain of hSos1 was analyzed by comparing the in vitro binding of HF to the PH domains of Ras-GRF1 (PH2 domain) (1Boguski M. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar) and PKD (22Waldron R.T. Iglesias T. Rozengurt E. J. Biol. Chem. 1999; 274: 9224-9230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To this end, the purified 6xHis-HF peptide was incubated with similar amounts of purified GST-PH (hSos1), GST-PH2 (Ras-GRF1), or GST-PH (PKD) proteins coupled to glutathione-Sepharose beads; and the bound proteins were detected as described above (Fig. 5 B). The 6xHis-HF peptide was found to bind only to GST-PH (hSos1) but not to GST-PH2 (Ras-GRF1) or GST-PH (PKD) proteins. To extrapolate these results to an in vivo situation, we carried out transient cotransfections of COS1 cells with the plasmids pCEFL-KZ-HA-NDP (coding for the epitope-tagged (HA) NDP region of hSos1), together with either pCEFL-KZ-AU5-HF (coding for the epitope-tagged (AU5) HF motif of hSos1), or pCEFL-KZ-AU5 (as negative control). Cellular lysates and anti-AU5 immunoprecipitates obtained under starving or stimulated cellular conditions were further analyzed by immunoblotting with anti-HA antibodies. We consistently detected HA-NDP coimmunoprecipitated with AU5-HF (Fig.6). The immunoblot analyses with anti-HA demonstrated that HA-NDP is associated with AU5-HF, mainly under stimulated conditions (Fig. 6). Similar results were observed in 293T cells (data not shown). All of these results suggested that the NDP region of hSos1 may establish in vivo stable complexes with the HF motif, depending on mitogenic conditions. The amino-terminal region of hSos1 (NDP region) contains DH and PH domains involved in Rac1 activation (5Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (390) Google Scholar) and phospholipid binding, respectively. In addition, it also contains a small region (residues 1–200) upstream of DH displaying high sequence similarity with histone H2A (24Baxevanis A.D. Arents G. Moudrianakis E.N. Landsman D. Nucleic Acids Res. 1995; 23: 2685-2691Crossref PubMed Scopus (182) Google Scholar), which we denoted here as the HF motif. Ectopic over-expression of the NDP region and its domains (HF, DH-PH, and DH) in mammalian cells blocks Ras, MAPK a

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