JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M005327200 on August 8, 2000

J. Biol. Chem., Vol. 275, Issue 45, 34901-34908, November 10, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/45/34901    most recent
M005327200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rebhun, J. F.
Right arrow Articles by Quilliam, L. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rebhun, J. F.
Right arrow Articles by Quilliam, L. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification of Guanine Nucleotide Exchange Factors (GEFs) for the Rap1 GTPase

REGULATION OF MR-GEF BY M-Ras-GTP INTERACTION*

John F. RebhunDagger, Ariel F. CastroDagger, and Lawrence A. Quilliam§

From the Department of Biochemistry and Molecular Biology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202

Received for publication, June 20, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the Ras subfamily of GTPases consists of ~20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ras is the prototype for a large superfamily of GTPases that regulate multiple cellular processes (1, 2). These include intracellular signal transduction for cell growth and differentiation (Ras subfamily), regulation of the actin cytoskeleton (Rho subfamily), membrane trafficking (Rab subfamily), vesicle trafficking (ARF subfamily), and nuclear transport (Ran) (1, 2). Each protein functions as a GTP/GDP-regulated switch that cycles between inactive GDP- and active GTP-bound states. This cycle is tightly controlled in vivo by two classes of regulatory proteins. Guanine nucleotide exchange factors (GEFs)1 serve as Ras activators by promoting acquisition of the active GTP-bound state and are the key link between cell surface receptors and Ras activation (3). Meanwhile, GTPase-activating proteins promote rapid GTP hydrolysis, returning Ras back to its inactive GDP-bound state (4).

Presently ~20 members of the Ras subfamily of GTPases have been identified in mammalian cells that include R-Ras, TC21/R-Ras2, M-Ras/R-Ras3, Rap1A, -1B, -2A, and -2B, Ral A and B, Rit, Rin, Dex-Ras, Rheb, Rhes, NOEY2, kappa B-Ras 1 and 2, as well as the classic (Ha-, K-, and N-) Ras proteins (1). Rap1A (Krev-1) was originally described as a competitive antagonist of K-Ras-induced transformation (5). The ability of Rap1 to block transformation is likely due to its ability to form non-productive complexes with Ras effectors such as c-Raf-1 (6). Although it fails to activate c-Raf-1, the Raf isomer, B-Raf is a major known effector of Rap1 and has been shown to elicit the ability of Rap1 to promote ERK/MAPK activation in PC12 cells as well as the LNCaP prostate carcinoma cell line (7, 8). Other putative Rap1 effectors include AF6 (9), RalGDS (10), and Krit1 (11), a molecule recently implicated in the formation of cavernous angiomas (12, 13).

M-Ras/R-Ras3 was identified as a Ras-related sequence in the EST data base (14-16) and independently by others (17, 18) due to its expression in muscle cells and interleukin 9-induced T helper cells. M-Ras is highly abundant in brain but is also expressed in other tissues and a broad variety of cultured cell lines (14-16). Although it is activated by Ras GEFs and can bind/activate some Ras protein effectors such as AF-6 and the Raf/ERK cascade (14, 15), unique sequences surrounding its effector binding domain suggest that, like other Ras-related GTPases, it will interact with novel downstream targets. Despite this prediction none have been identified so far.

Although over 20 Ras family GTPases have been identified, only a handful of the GEFs that regulate them have been described as follows: Sos, GRF and GRP isoforms for Ras, the RalGDS family for Ral, and C3G, RapGRP/CalDAG GEF, Epac/cAMP-GEFs for Rap1 (3, 19-21). One unique GEF, Smg GDS (small molecular weight GTP-binding protein guanine nucleotide dissociation stimulator), catalyzes exchange on Rho family GTPases as well as Rap1 and K-Ras in vitro (22, 23). Smg GDS is composed of 11 armadillo repeats similar to those found in APC and catenins (24) and does not share significant homology with CDC25 (25).

The catalytic domains of all other Ras subfamily GEFs share ~30% homology with each other and the Saccharomyces cerevisiae protein, CDC25. Conservation between "CDC25 homology" domains is greatest within structurally conserved regions (SCR) 1-3 that were first noted by Boguski and McCormick (25), whereas additional C-terminal regions (SCR 4 and 5) have subsequently become evident (Fig. 1) (26). A region outside of the core catalytic domain, referred to as Ras exchanger motif (REM), conserved non-catalytic, or SCR 0 has also been noted (27-29). Based on the Sos1 Ras GEF x-ray crystal structure, REM/SCR 0 is a structural component that binds to SCR4 and is not involved in Ras interaction (29).

Besides the common "CDC25 homology" catalytic domain, GEFs possess a wide variety of domains that are important for the regulation of their function. For instance, Sos contains proline-rich clusters that interact with the SH3 domains of adapter proteins such as Grb2 bringing Sos to the membrane after receptor tyrosine kinase activation (3). Translocation to the membrane is assumed to be critical in the activation of GEFs since it brings them into contact with membrane-bound GTPases. PH domains are also found in several GEFs, including Sos and GRFs, where this domain is also important for membrane interaction (30). The GRP or CalDAG GEFs for Ras and Rap each possess EF hands and C1 domains and are regulated by calcium and/or DAG (19, 20, 31). Furthermore, the Rap GEFs, Epac/cAMP-GEF 1 and 2, are regulated by cyclic AMP (21, 32) indicating that Ras function can be regulated by receptors others than tyrosine kinases. Finally, binding to Ras activates the RalGDS family of Ral GEFs (1). These proteins contain a Ras-associating or RalGDS/AF6-homology (RA) domain responsible for the interaction with Ras-GTP (33, 34).

Since the increasing number of Ras family members suggested the existence of additional Ras GEFs to regulate them, we searched the NCBI data bases for novel CDC25 homology domain-containing proteins. Three of the putative GEFs that we identified promote the formation of Rap1-GTP in vivo, and splice variants of a fourth activate Ral (26). Two of the Rap GEFs contain RA domains. Just as the Ral family GEFs, RalGDS, RGL and RGL2/Rlf, are downstream effectors of Ras, one of the novel GEFs, MR-GEF (M-Ras-regulated Rap GEF), appears to be regulated by interaction with M-Ras in a GTP-dependent manner. Since Rap1 can antagonize the activation of Raf-1 by other GTPases, negative regulation of Rap1 by M-Ras via MR-GEF may help facilitate M-Ras-induced activation of Raf-1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructs-- Full-length human MR-GEF (KIAA0277), PDZ-GEF (KIAA0313), and GRP3 (KIAA0846) were kindly provided by Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan). MR-GEF was digested with BglII and subcloned into the BamHI site of pcDNA3 vector. ClaI and BamHI sites were incorporated immediately 5' of the presumed start codon of PDZ-GEF, MR-GEF, and GRP3 by polymerase chain reaction. A ClaI/NheI fragment of PDZ-GEF and a ClaI/XbaI fragment of MR-GEF from pcDNA3 were inserted into the ClaI/XbaI site of pFLAG-CMV2 vector (Eastman Kodak Co.). The EcoRV/XbaI fragment of GRP3 was subcloned into pcDNA3. Smg GDS was isolated as described previously (23) and subcloned into the BamHI site of pFLAG-CMV2. Ral, Rap2B, and Rheb were provided by D. Andres (University of Kentucky), J. de Gunzburg (Institut Curie, Paris, France), and G. Clark (NCI, National Institutes of Health), respectively. Rlf and the pGEX-RID (Ral interacting domain from Ral BP1, residues 648-778) were provided by D. Andres. C3G was obtained from S. Tanaka and H. Hanafusa (Rockefeller University).

Miscellaneous-- A 1.3-kb EcoRI fragment of MR-GEF (residues 0-1354) and a 1.0-kb BglII fragment of PDZ-GEF (residues 1571-2576) were radiolabeled with [32P]dCTP and used to probe human multitissue Northern blots (CLONTECH) as described previously (26). 293T human embryonic kidney cells were cultured as described (26). GST-Ras family GTPase fusion proteins and GST-RalGDS/AF6 binding domains were produced as described previously (26). Luciferase assays were performed by co-transfection of LNCaP cells with 0.125 µg of Gal4-Elk, 1.25 µg of 5XGal4-Luc, and 1 µg of pcDNA3 encoding the indicated gene, using Fugene6 (Roche Molecular Biochemicals) (14). After 24 h, cells were serum-starved overnight, and luciferase activity was determined as described.

Ras Protein Binding Assay-- This assay was completed as described previously (26). Briefly, 293T cells were transfected with vectors encoding FLAG-MR-GEF, FLAG-PDZ-GEF, or FLAG-GRP3 using NovaFECTOR (Venn Nova Inc.). After 48 h, cells were lysed and mixed with ~20 µg of GST-Ras fusion proteins bound to glutathione-agarose beads. This slurry was tumbled for 2 h at 4 °C, and then the beads were washed 4 times with Ras buffer (20 mM Tris-HCl, pH 7.6, 5% glycerol, 50 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and 1 mM dithiothreitol), and the bound GEFs were visualized by SDS-PAGE immunoblotting. For the assay of MR-GEF or PDZ-GEF as Ras effectors, the glutathione-agarose-immobilized GST-Ras proteins (~10 µg) were loaded with 100 µM GDP or GTPgamma S and the 293T cell lysates expressing FLAG-tagged MR-GEF, MR-GEFDelta cat, or PDZ-GEF were prepared with Ras buffer containing 5 mM MgCl2 instead of EDTA.

Non-radioactive in Vivo Exchange Assay-- 293T cells were transfected with 750 ng of pFLAG-CMV2-Rap1A, RalA, or Ha-Ras and 750 ng of the indicated GEF using 8 µl of NovaFECTOR/60-mm dish. After 24 h, serum was removed for another 18 h. Cells were rinsed twice in phosphate-buffered saline, lysed with Ral buffer (0.5 ml/dish; 50 mM Tris-HCl, pH 7.4, 10% glycerol, 200 mM NaCl, 2.5 mM MgCl2, 1% IGEPAL (Sigma), aprotinin (0.05 TIU/ml), 1 mM PMSF), and cleared by centrifugation for 10 min at 12,000 × g. Approximately 10 µg of GST-Raf (residues 2-140; for Rap1A and Ha-Ras) or -RID (residues 648-778; for RalA) bound to 35 µl of glutathione-agarose beads (50% slurry) were rinsed twice with Ral buffer and then tumbled with 400 µl of cell lysate for 1 h at 4 °C. The beads were washed 4 times with Ral buffer, omitting protease inhibitors. The activated FLAG-tagged GTPase bound to the GST-RA domain were visualized by immunoblotting.

Radioactive in Vivo Exchange Assay-- 293T cells were transfected with 750 ng of pFLAG-CMV2-Rap and increasing amounts of pcDNA3 MR-GEF with and without 500 ng of M-Ras71L using NovaFECTOR (empty vector was used to equalize the amount of transfected DNA). After 18 h the media were changed to serum-free media, and the cells were incubated for another 20 h. The media were changed to phosphate-free and serum-free media for 30 min, and then 150 µCi of [32P]orthophosphate was added to each dish. After 2 h of incubation the cells were lysed in 500 µl of lysis buffer (50 mM Hepes, pH 7.5, 500 mM NaCl, 6 mM MgCl2, 1% Triton X-100, 0.5% deoxycholate, 0.05% SDS, 1 mM EDTA, 1 mM PMSF, and aprotinin (0.05 TIU/ml)), cleared by centrifugation for 5 min at 12,000 × g, and then precleared for 15 min at 4 °C with 30 µl of protein A/G-agarose (Santa Cruz Biotechnology, Inc.). The precleared lysate was tumbled with 10 µg of M2 anti-FLAG antibody (Sigma) for 30 min at 4 °C and then incubated with 30 µl of protein A/G-agarose at 4 °C. Bound proteins were washed 6 times with ice-cold Wash buffer (50 mM Hepes, pH 7.5, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and 0.005% SDS). Twenty µl of Elution buffer (2 mM EDTA, 5 mM dithiothreitol, 1 mM GDP, 1 mM GTP, and 0.2% SDS) were added to the beads which were incubated for 20 min at 68 °C. Ten µl of the supernatant were spotted on polyethyleneimine-cellulose TLC plates (Bakerflex, Inc.) and run in 0.75 M KH2PO4. GDP and GTP spots were quantitated with a beta -scanner (Ambis, Inc.), and percent GTP was calculated as (GTP cpm/(GDP cpm × 1.5) + GTP cpm) × 100. Levels of MR-GEF expression were determined by immunoblotting with an MR-GEF-specific rabbit polyclonal antibody raised against residues 1-77. Bands of MR-GEF were quantitated using ECL reagents and Scion Image version 1.6c.

Co-immunoprecipitation-- 293T cells were co-transfected with 2 µg of either pFLAG-CMV2-MR-GEF or pFLAG-CMV2-MR-GEFDelta cat and 2 µg of either pCGN-M-ras(wt), -M-ras(71L), -Ha-ras(61L), or R-ras(87L) using 20 µl of NovaFECTOR/100-mm dish. After 48 h the cells were lysed in 1 ml of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1% IGEPAL, 1 mM EGTA, 100 mM NaF, 10% glycerol, 1 mM PMSF, and aprotinin (0.05 TIU/ml)). Cellular debris was removed by a 3-min, 13,000 × g spin, and the cellular lysate was precleared for 15 min at 4 °C with 30 µl of protein A/G-agarose. The precleared lysate was tumbled with 5 µg of anti-HA antibody (Babco) for 2 h at 4 °C followed by an additional hour with 30 µl of protein A/G-agarose and beads were washed 5 times with lysis buffer. Co-precipitation of either MR-GEF or MR-GEFDelta cat with Ras proteins was visualized by immunoblotting with anti-FLAG antibody (M2; Sigma) following SDS-PAGE. Levels of Ras protein expression and precipitation were determined by HA antibody using ECL reagents (Amersham Pharmacia Biotech).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Tissue Distribution of Multiple Putative Novel GEF cDNAs-- Due to the relatively small number of CDC25 homology domain-containing family GEFs currently existing to regulate the Ras subfamily of GTPases, we used the SCR2 domain of GRF1 to search the NCBI-expressed sequence tag and non-redundant data bases for additional family members. We then re-screened the data bases using the isolated sequences to identify further cDNAs. By using this approach, we identified multiple hits. These included the following: 1) a cAMP-activated GEF, Epac, subsequently described by others (21, 32); 2) GRP3 that closely resembles the Ca2+/diacylglycerol-activated Ras GEF, GRP1, and the related Rap1 GEF, GRP2 (19, 20, 31) (Fig. 1); 3) MR-GEF shared strongest homology with the cAMP-activated Rap GEF Epac; and 4) another novel putative GEF, PDZ-GEF. Characterization of a novel family of Ral GEFs, RalGPS, has been described elsewhere (26). As seen in Fig. 1, all of these GEFs shared sequence homology in REM/SCR 0, SCR 1-3, as well as two other regions that are boxed and referred to here as SCR 4 and 5. SCR 4 contains a highly conserved I/VNF motif that sits in a hydrophobic groove formed by SCR 0 (29), and SCR 5 represents a highly conserved C-terminal sequence. GRP3 mRNA is ubiquitously expressed and is most abundant in kidney, brain, lung, and heart (HUGE protein data base, Kazusa DNA Research Institute, Chiba, Japan). To determine the tissue distribution of MR-GEF and PDZ-GEF, cDNAs were used to probe a multitissue Northern blot. As shown in Fig. 2, both GEF messages were most abundantly expressed in brain with detectable levels in various other tissues, many of epithelial origin.


View larger version (80K):
[in this window]
[in a new window]
  		Fig. 1.   Structure and alignment of Rap1 exchanges factors. A, model of Rap1 exchange factor open reading frames showing the position of the catalytic domain, RA domain, PDZ domain, REM (Scr 0) domain, cyclic nucleotide-binding domain, proline-rich region, EF hands, and diacylglycerol/phorbol ester-binding domain. MR-GEFDelta cat is a truncated construct used in Figs. 5 and 6. B, sequence alignment of catalytic domains from Rap GEFs of human MR-GEF (GenBankTM accession number D87467), PDZ-GEF (GenBankTM accession number AB002311), Epac (GenBankTM accession number AF103905), GRP3 (GenBankTM accession number AB020653), GRP2 (GenBankTM accession number U78170), GRP1 (GenBankTM accession number AF106071), C3G (GenBankTM accession number D21239), and the Ras GEFs Sos1 (GenBankTM accession number L13857), and rat GRF1 (GenBankTM accession number X67241). C, sequence alignment of RalGDS/AF-6 association domains of mouse Nore1 (GenBankTM accession number AF053959) and human Rgl (GenBankTM accession number U68142), AF-6 N-terminal domain (GenBankTM accession number U02478), PDZ-GEF, MR-GEF, Link GEFII (AF117946), and Epac 2 (cAMP-GEFII; U78516).


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 2.   Multitissue Northern blot showing MR-GEF and PDZ-GEF distribution. An ~1.3-kb EcoRI fragment encompassing the N terminus of MR-GEF coding region and a ~1.0-kb BglII fragment of PDZ-GEF coding region were used to screen a human multitissue Northern blot essentially as described by the manufacturer. skel. muscle, skeletal muscle; sm. intestine, small intestine.

MR-GEF and PDZ-GEF Are Rap Family Exchange Factors-- Since the above sequence alignment predicted that MR-GEF and PDZ-GEF should act as GEFs for Ras family GTPases, we determined their substrate specificity. To do this we initially took advantage of the fact that GEFs bind with high affinity to nucleotide-free GTP-binding proteins (35). Following the expression of FLAG epitope-tagged GEFs in 293T cells, nucleotide-free GST-Rap1A was found to precipitate both GEFs from cell lysates (Fig. 3A). GST-Rap2B also precipitated PDZ-GEF and weakly MR-GEF suggesting that these GEFs might be Rap-specific exchange factors (Fig. 3A). There was no significant interaction with other GTPases indicating that they were unlikely to be MR-GEF and PDZ-GEF substrates. In contrast, GRP3 bound to all GTPases tested in a nonspecific manner.2


View larger version (58K):
[in this window]
[in a new window]
  		Fig. 3.   MR-GEF, PDZ-GEF, and GRP3 are Rap GEFs. A, the indicated GST-Ras fusion proteins (20 µg) were stripped of nucleotide and used to precipitate FLAG-tagged MR-GEF and PDZ-GEF from 293T cell lysates. Bound GEFs were detected by Western blotting precipitates with M2 anti-FLAG antibody. B, empty vector or vectors encoding C3G, MR-GEF, PDZ-GEF, GRP3, Smg GDS, or Rlf were co-transfected into 293T cells with vectors encoding FLAG-tagged Rap1A, Ral A, or Ha-Ras as indicated. Following overnight starvation, cells were lysed, and Rap1A-GTP, Ral A-GTP, and Ha-Ras-GTP were extracted by incubation with the GTPase-binding domains of Raf1, Ral BP1, or Raf-1, respectively. Stimulation of Rap1A/Ral A/Ha-Ras-GTP accumulation was determined by Western blotting with anti-FLAG antibody. Lower bands indicate equal expression of GTPases in cell lysates. All data are representative of at least three independent experiments.

To confirm that the GEFs that bound a particular Ras protein in vitro also induced nucleotide exchange in vivo, we transiently co-expressed various GEFs along with epitope-tagged, Rap1A, Ral A, and Ha-Ras proteins in 293T cells and, following overnight serum-starvation, measured the formation of Ras-GTP. To do this we took advantage of the fact that the Ras/Rap1 effector Raf and the Ral target Ral BP1 have a much higher affinity for GTP- versus GDP-bound GTPases (36). A GST fusion protein containing the Rap1-binding domain of Raf was used to precipitate FLAG-tagged Rap1A-GTP from cell lysates. As shown in Fig. 3B, C3G (a known Rap1 GEF), MR-GEF, and PDZ-GEF all promoted the accumulation of Rap-GTP but not Ha-Ras- or RalA-GTP. In contrast, Rlf activated RalA but not Rap1A under similar conditions. Preliminary data indicate that PDZ-GEF can also activate Rap2.2 As predicted from previous in vitro studies, bovine Smg GDS activated Rap1 but not Ha-Ras. It also promoted in vivo Ral A activation. Only the putative GEF GRP3 that shares significant homology with the diacylglycerol and Ca2+-activated GRP1 and -2 (19, 20) elevated Ha-Ras GTP levels. These data suggest that each putative GEF is functionally active and that multiple GEFs exist to regulate Rap1 in vivo.

Rap GEFs Can Induce Elk-1 Activation in LNCaP Cells-- Since Rap1 is required for cyclic AMP-induced B-Raf/ERK activation in PC12 and LNCaP cells (7, 8), we next examined whether various Rap GEFs could mimic Rap1 in the LNCaP prostate cancer cell line to promote downstream Elk-1 activation. As seen in Fig. 4, overexpression of MR-GEF, PDZ-GEF, GRP3, C3G, or Smg GDS to varying degrees mimicked the ability of activated Rap1A(63E) to promote Elk-1-induced luciferase expression. These studies demonstrate that the novel Rap1 exchange factors are biologically active following exogenous expression. We presume that the greater activation response of Elk-1 to GRP3 than other Rap GEFs is due to its dual role as both a Ras and Rap1 GEF.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Rap exchange factors activate Elk-1 in the LNCaP cell line. LNCaP cells were transfected with Gal4-Elk and 5XGal4-Luc luciferase reporter constructs along with plasmids encoding activated Rap1A or the indicated exchange factors. After 24 h, cells were serum-starved overnight, and luciferase activity from cell lysates was determined. Rap1 and each of the GEFs were found to promote Elk-1 activation. Data are mean ± range for duplicate plates and are representative of at least three experiments.

M-Ras-GTP Associates with MR-GEF-- The subcellular localization and activity of previously characterized GEFs are modulated by various regulatory domains. Examination of Ras family GEFs indicated that MR-GEF and PDZ-GEF both contain a region immediately N-terminal to SCR 1 that shares sequence homology with the RA domains of RalGDS and AF6 (Fig. 1C). These domains interact with the effector-binding loop of active GTP-bound Ras proteins (37). To determine if the putative RA domains in MR-GEF and PDZ-GEF interact with Ras proteins in vitro, GST-Ras fusion proteins loaded with the non-hydrolyzable GTP analog, GTPgamma S, were incubated in the presence of millimolar MgCl2 with 293T cell lysates containing FLAG-tagged MR-GEF or PDZ-GEF. In the presence of MgGTP there should be no significant association of GTPases with GEF catalytic domains (38). As shown in Fig. 5A, PDZ-GEF specifically bound to Rap1A and Rap2B. Since Rap proteins did not bind to C3G or MR-GEF under similar conditions, in the presence of GTP, the interaction of PDZ-GEF with Rap1A would appear to be via its RA rather than catalytic domain.


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 5.   MR-GEF and PDZ-GEF behave as Ras-GTPase effectors. A, GST-Ras proteins were loaded with GTPgamma S and used to precipitate FLAG-tagged MR-GEF or PDZ-GEF from 293T cell lysates in the presence of MgCl2. Bound GEFs were detected by Western blotting with M2 anti-FLAG antibody. B, in vitro interaction of MR-GEF with M-Ras was tested as in A except that GST-M-Ras was loaded with either GDP or GTPgamma S as indicated. C, GTP-dependent interaction of M-Ras with the N-terminal, non-catalytic, region of MR-GEF (MR-GEFDelta cat, see Fig. 1A) was performed as in B. Nucleotide-free Rap1 was used as a control to demonstrate that the truncated MR-GEF protein could no longer bind its substrate. The experiments in A-C were performed at least three times, giving similar results.

The RA domain of MR-GEF is not as highly conserved with those of RalGDS and AF6 as is PDZ-GEF (see Fig. 1C). However, MR-GEF was found to associate specifically with M-Ras (Fig. 5A). This in vitro interaction occurred in a GTP-dependent manner (Fig. 5B) and was not a result of M-Ras binding to the catalytic domain since an N-terminal fragment, MR-GEFDelta cat truncated in SCR 1 (see Fig. 1A), retained its ability to bind M-Ras-GTP (Fig. 5C). This N-terminal region encompasses the RA domain of MR-GEF.

In Vivo Association with M-Ras Inhibited MR-GEF-induced Rap1 Activation-- Overall, the in vitro experiments implied that MR-GEF is a specific M-Ras effector. Hence, we tested whether M-Ras could interact with MR-GEF in vivo. Following co-expression in 293T cells, MR-GEF co-precipitated with constitutively activated M-Ras(71L) (Fig. 6A). In contrast, the predominantly GDP-bound M-Ras(wt) could not efficiently precipitate MR-GEF demonstrating that the co-precipitation of M-Ras and MR-GEF in vivo is GTP-dependent. Additionally, MR-GEF failed to interact with activated Ha-Ras(61L) or R-Ras(87L), again suggesting that the interaction with M-Ras is specific. As seen in vitro, M-Ras(71L) associated in vivo with MR-GEFDelta cat (Fig. 6B). Thus, the N-terminal region of MR-GEF is responsible for the in vivo interaction with M-Ras-GTP and it may serve as a regulatory region of MR-GEF function.


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 6.   In vivo interaction of activated M-Ras with MR-GEF. A, HA-tagged M-Ras(wt) or activated mutants of M-, Ha-, or R-Ras were co-expressed with FLAG-tagged MR-GEF in 293T cells. Following immunoprecipitation of Ha-Ras proteins, co-precipitation (Co-IP) of MR-GEF was determined by Western blotting with M2 anti-FLAG antibody. B, co-immunoprecipitation study of MR-GEFDelta cat was carried out as in A, indicating that the N terminus of MR-GEF is sufficient for M-Ras binding. Only interaction with activated mutants of M- or Ha-Ras was tested. Results shown are representatives of at least three independent experiments.

Since binding of Ras-GTP to RalGDS results in activation of Ral (39), we examined whether M-Ras associates with MR-GEF in vivo to modulate its ability to activate Rap1. As seen in Fig. 7A, overexpression of the active M-Ras(71L) mutant in the absence of MR-GEF had no significant effect on Rap1-GTP levels in vivo. However, it suppressed the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. Because the presence of M-Ras(71L) elevated the expression of MR-GEF from its CMV promoter,2 subsequent experiments were normalized for MR-GEF expression level. As shown in Fig. 7B, after such normalization, M-Ras-GTP was found to inhibit MR-GEF-induced Rap1A activation by over 50%. These results suggest that M-Ras is a negative regulator of Rap1 function. Whether this inhibition is due to subcellular sequestration of MR-GEF and/or the direct regulation of Rap GEF activity by M-Ras binding to the RA domain will require the generation of active full-length recombinant MR-GEF protein to perform in vitro exchange assays.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7.   Inhibition of MR-GEF activity by M-Ras(71L) in vivo. A, 293T cells were co-transfected with plasmid encoding FLAG-tagged Rap1A (750 ng/dish) and the indicated amounts of pcDNA3 M-ras(71L) ± 300 ng of pcDNA3 MR-GEF/dish. An in vivo exchange assay was then performed as described under "Experimental Procedures" using the GST-Raf-(2-140) to isolate Rap1-GTP. Data are representative of five similar experiments. B, the ability of M-Ras(71L) (500 ng plasmid) to inhibit MR-GEF-induced Rap1A activation was further determined at three different GEF plasmid concentrations. Data were normalized for MR-GEF concentration and expressed as a percentage of Rap1A stimulation in the presence of empty vector (absence of M-Ras). MR-GEF levels in the cell lysates were determined by Western blotting with specific polyclonal antiserum, and beta -actin levels were measured on the same blot to control for total protein harvested. Data are mean ± S.D. and are representative of three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Currently 14 members of the Rho family of GTPase have been identified for which there are more than 35 GEFs (the Dbl/PH proteins) responsible for loading them with GTP (40). Although there are an increasing number of Ras family members, until recently only a limited number of CDC25 homology domain-containing GEFs existed to mediate Ras protein activation. A sequence homology search of the DNA data bases has now revealed several additional members. We report here that putative exchange factors MR-GEF and PDZ-GEF activate Rap1, and a third, GRP3, activates Rap and Ras. An additional Ral GEF family "RalGPS" has been described elsewhere (26). We found that MR-GEF and PDZ-GEF contain RA domains. Importantly, the RA domain present in MR-GEF is regulated by interaction with M-Ras in a GTP-dependent manner and is conserved in other Rap GEFs. In agreement with the results presented here, several other recent reports have described these new GEFs (41-47). However, this study is the first to report the interaction of activated M-Ras with MR-GEF and to demonstrate M-Ras regulation of the Rap1 exchange activity of MR-GEF.

The identification of the above proteins brings the total number of known Rap1 GEFs to nine (SmgGDS, C3G, GRP2 and -3, Epac 1 and 2, MR-GEF, PDZ-GEFs 1 and 2). Within the group of Rap GEFs, Smg GDS presents unusual characteristics. It does not possess the CDC25 homology domain found in other Ras GEFs and has previously been reported to activate Rap1, Rac, Rho, and K-Ras in vitro (22, 23). Here we showed that Smg GDS acts as a Rap GEF in vivo and further demonstrate its ability to activate Ral. This is consistent with the observed interaction of Xenopus Ral with a frog Smg GDS-related protein in a yeast two-hybrid screen (48) and the presence of a polybasic C terminus in Ral similar to that found in other Smg GDS substrates. Why a single GEF is responsible for activating so many GTPases and how it is regulated will require further investigation. However, our in vivo studies confirm previous in vitro and functional assays that suggested that Smg GDS would act as a Rap1 GEF.

It is interesting to note how divergent the mechanisms of Rap GEF regulation are. C3G is activated downstream of tyrosine kinases, being recruited by adapter proteins such as Crk and p130Cas (49). SHEP1/NSP3, a putative GEF that can bind Rap1 in vitro, is also downstream of protein tyrosine kinases (50, 51). Epac/cAMP-GEFs 1 and 2 are activated by the second messenger cAMP, whereas GRP2 (and potentially GRP3) is activated by phorbol esters/diacylglycerol and/or calcium (19-21, 32). The discovery of PDZ-GEF and MR-GEF identifies novel domains/mechanisms to direct Rap GEF activation. PDZ-GEF has a number of putative domains that may regulate its activity. The PDZ domain regulates the attachment of PDZ-GEFs to the plasma membrane of HEK cells (47), whereas a putative C-terminal (SAV) PDZ-binding motif may be involved in the interaction of PDZ-GEF with a six PDZ domain-containing synaptic scaffolding protein, S-SCAM, described by Takai and colleagues (44).

PDZ-GEF also possesses a cAMP-binding domain, sharing weak homology with those of Epac and the protein kinase A regulatory subunits (21, 32). The binding of cAMP to PDZ-GEF is not required for its activation of Rap, although the presence of the cAMP-binding domain does have a slight inhibitory role (41), analogous to the domain in Epac, which cannot activate Rap1A unless cAMP is present (21). During the completion of this manuscript it was reported that the presence of cAMP or cGMP allowed PDZ-GEF to activate Ha-Ras in vivo without affecting Rap1 activation (47). We saw no binding of PDZ-GEF to Ha-Ras in vitro and no activation of Ha-Ras by PDZ-GEF in the absence of cAMP in vivo, suggesting that Ha-Ras activation by PDZ-GEF is highly dependent on cyclic nucleotides and that they must act by permitting/promoting Ha-Ras-GEF association. PDZ-GEF is not the only example of a Rap1 GEF that also works on Ras. We found that GRP3 also activates both Rap1 and Ha-Ras and is consistent with reports indicating that Ras and Rap1 are concomitantly activated by mitogens (10, 52). Like GRP1 and GRP2, GRP3 contains EF hands and a C1 domain suggesting that it is regulated by calcium and/or diacylglycerol. Although we have seen strong activation of Ha-Ras and Rap1 by GRP3 under serum-starved conditions, it will be interesting to study whether activation by calcium and/or diacylglycerol regulates the specificity of GRP3 for Ha-Ras and Rap1 as cAMP appears to do with PDZ-GEF.

The presence of RA domains in PDZ-GEF and MR-GEF suggests a novel mechanism of regulating the activity and/or specificity of Rap GEFs. The binding of Rap1 or -2 to the RA domain of PDZ-GEF may modulate its activity as part of a regulatory feedback mechanism. To date, regulation of PDZ-GEF activity by Rap1-GTP has not been reported. The C terminus of the MR-GEF RA domain is poorly conserved with the "classical" RA domains of RalGDS and AF-6 (Fig. 1C). However, the ability of M-Ras to bind the N-terminal regulatory domain of MR-GEF in a GTP-dependent manner together with its ability to inhibit the activation of Rap1 by MR-GEF strongly suggest that association with the RA domain inhibits MR-GEF activity. Additionally, it was interesting to find the putative RA domain of MR-GEF is conserved in two other GEFs. In fact, the Rap1 GEF Epac2 and the putative exchange factor Link GEFII contain considerable homology to MR-GEF beyond their RA and CDC25 homology domains (Fig. 1C). This suggests that there is a family of Rap GEFs sensitive to M-Ras or other Ras protein regulations. As mentioned above, association of M-Ras with MR-GEF appears to inhibit Rap activation. Such negative regulation seems logical given the previous studies on these two GTPases. M-Ras, like the classical Ras proteins, induces cellular transformation of 3T3 fibroblasts (14, 15), whereas Rap1 was initially discovered as a suppressor of K-Ras-induced transformation, thus antagonizing Ras functions. It has similarly been shown to antagonize T cell activation (53). Under this scenario, it is reasonable to speculate that activation of M-Ras could be responsible for the negative regulation of the Rap1 pathway through its association with the RA domain of Rap GEFs. The lack of activated Rap1 would lead to the disassociation of the inactive Rap-Raf-1 complex leaving Raf-1 free for activation by M-Ras or other Ras proteins. As M-Ras is widely expressed and in some tissues is present at higher levels than classical Ras proteins (16), such a mechanism may apply to several systems where Rap-Ras pathways result in different physiological outcomes. Currently, this hypothesis is under investigation.

Regulation of downstream GTPases by Ras was originally described for a family of Ral exchange factors (RalGDS) that contain C-terminal RA domains (34). This interaction serves to recruit Ral GEFs to the membrane where they contact and activate the Ral GTPase. It is interesting to note that MR-GEF and PDZ-GEF have RA domains immediately N-terminal to their catalytic domains. Such insertions of regulatory domains within their catalytic domains may be a means of modulating the activity as well as the location of the catalytic domain. Although the inhibitory role of M-Ras in Rap1 activation by MR-GEF supports this idea, further studies will be required to clarify their role. It will also be interesting to determine whether the Rap1/2 interaction with the RA domain of PDZ-GEF results in a similar effect. MR-GEF and PDZ-GEF join a growing list of proteins containing putative Ras associating domains for which the physiological relevance/function remains to be established (54, 55).

In contrast to the Ras/Rap1 antagonism described above, Rap1 can activate ERK in certain cell types (7, 8). This activation is dependent on the presence of the Rap1 effector B-Raf, which can activate the ERK pathway leading to downstream Elk1 activation. We have shown here that overexpression of Rap GEFs can promote Elk1-induced gene expression in LNCaP cells. Multiple agents that signal via growth factor receptors, cAMP or Ca2+, have been reported to promote the growth of prostate cells (56, 57). Furthermore, Chen et al. (8) have reported that epidermal growth factor and cAMP can cooperate to activate ERK in a Rap1-dependent manner. Since GEFs are the major link between receptors and Ras protein activation, it will be interesting to determine which of the various Rap GEFs are responsible for prostate cell growth.

In conclusion, we have identified several Ras family GEFs and demonstrated their ability to activate Rap1 and/or Ras in vivo and subsequently activate the downstream MAPK cascade when overexpressed in prostate cancer cells. One of these GEFs MR-GEF contains an RA domain that is well conserved in two additional Rap GEFs. We have shown that MR-GEF behaves as a specific M-Ras effector and can couple this GTPase to the inhibition of Rap1 activation. These results suggest a second GTPase regulatory connection analogous to the Ras-RalGDS-Ral pathway that may help to understand the physiological function of M-Ras as well as the role of Rap1 in Ras signaling.

    ACKNOWLEDGEMENT

We thank Chen Bi for technical assistance.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RPG-00-125-01-TBE.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Both authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS-4075, Indianapolis, IN 46202. Tel.: 317-274-8550; Fax: 317-274-4686; E-mail: lquillia@iupui.edu.

Published, JBC Papers in Press, August 8, 2000, DOI 10.1074/jbc.M005327200

2 J. F. Rebhun, A. F. Castro, and L. A. Quilliam, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GEF, guanine nucleotide exchange factor; ERK, extracellular receptor activated kinase; MAPK, mitogen-activated protein kinase; RA domain, Ras-association or RalGDS/AF6 homology domain; TIU, trypsin inhibitory units; kb, kilobase pair; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; wt, wild type; HA, hemagglutinin; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; SCR, structurally conserved regions; REM, Ras exchanger motif; DAG, diacylglycerol; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J. (1998) Oncogene 17, 1395-1413
2. Macara, I. G., Lounsbury, K. M., Richards, S. A., McKiernan, C., and Bar-Sagi, D. (1996) FASEB J. 10, 625-630
3. Quilliam, L. A., Khosravi-Far, R., Huff, S. Y., and Der, C. J. (1995) BioEssays 17, 395-404
4. Wittinghofer, A. (1998) Biol. Chem. Hoppe-Seyler 379, 933-937
5. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84
6. Cook, S. J., Rubinfeld, B., Albert, I., and McCormick, F. (1993) EMBO J. 12, 3475-3485
7. Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. (1997) Cell 89, 73-82
8. Chen, T., Cho, R. W., Stork, P. J., and Weber, M. J. (1999) Cancer Res. 59, 213-218
9. Linnemann, T., Geyer, M., Jaitner, B. K., Block, C., Kalbitzer, H. R., Wittinghofer, A., and Herrmann, C. (1999) J. Biol. Chem. 274, 13556-13562
10. Zwartkruis, F. J., Wolthuis, R. M., Nabben, N. M., Franke, B., and Bos, J. L. (1998) EMBO J. 17, 5905-5912
11. Serebriiskii, I., Estojak, J., Sonoda, G., Testa, J. R., and Golemis, E. A. (1997) Oncogene 15, 1043-1049
12. Sahoo, T., Johnson, E. W., Thomas, J. W., Kuehl, P. M., Jones, T. L., Dokken, C. G., Touchman, J. W., Gallione, C. J., Lee-Lin, S. Q., Kosofsky, B., Kurth, J. H., Louis, D. N., Mettler, G., Morrison, L., Gil-Nagel, A., Rich, S. S., Zabramski, J. M., Boguski, M. S., Green, E. D., and Marchuk, D. A. (1999) Hum. Mol. Genet. 8, 2325-2333
13. Laberge-le Couteulx, S., Jung, H. H., Labauge, P., Houtteville, J. P., Lescoat, C., Cecillon, M., Marechal, E., Joutel, A., Bach, J. F., and Tournier-Lasserve, E. (1999) Nat. Genet. 23, 189-193
14. Quilliam, L. A., Graham, K. R., Castro, A. F., Martin, C. B., Der, C. J., and Bi, C. (1999) J. Biol. Chem. 274, 23850-23857
15. Kimmelman, A., Tolkacheva, T., Lorenzi, M. V., Osada, M., and Chan, A. M. (1997) Oncogene 15, 2675-2685
16. Ehrhardt, G. R., Leslie, K. B., Lee, F., Wieler, J. S., and Schrader, J. W. (1999) Blood 94, 2433-2444
17. Matsumoto, K., Asano, T., and Endo, T. (1997) Oncogene 15, 2409-2417
18. Louahed, J., Grasso, L., De Smet, C., Van Roost, E., Wildmann, C., Nicolaides, N. C., Levitt, R. C., and Renauld, J.-C. (1999) Blood 94, 1701-1710
19. Ebinu, J. O., Bottorff, D. A., Chan, E. Y., Stang, S. L., Dunn, R. J., and Stone, J. C. (1998) Science 280, 1082-1086
20. Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283
21. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Nature 396, 474-477
22. Mizuno, T., Kaibuchi, K., Yamamoto, T., Kawamura, M., Sakoda, T., Fujioka, H., Matsuura, Y., and Takai, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6442-6446
23. Chuang, T. H., Xu, X., Quilliam, L. A., and Bokoch, G. M. (1994) Biochem. J. 303, 761-767
24. Peifer, M., Berg, S., and Reynolds, A. B. (1994) Cell 76, 789-791
25. Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-654
26. Rebhun, J. F., Chen, H., and Quilliam, L. A. (2000) J. Biol. Chem. 275, 13406-13410
27. Lai, C. C., Boguski, M., Broek, D., and Powers, S. (1993) Mol. Cell. Biol. 13, 1345-1352
28. Fam, N. P., Fan, W. T., Wang, Z., Zhang, L. J., Chen, H., and Moran, M. F. (1997) Mol. Cell. Biol. 17, 1396-1406
29. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343
30. Chen, R. H., Corbalan-Garcia, S., and Bar-Sagi, D. (1997) EMBO J. 16, 1351-1359
31. Tognon, C. E., Kirk, H. E., Passmore, L. A., Whitehead, I. P., Der, C. J., and Kay, R. J. (1998) Mol. Cell. Biol. 18, 6995-7008
32. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Science 282, 2275-2279
33. Ponting, C. P., and Benjamin, D. R. (1996) Trends Biochem. Sci. 21, 422-425
34. Hofer, F., Fields, S., Schneider, C., and Martin, G. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11089-11093
35. Feig, L. A. (1999) Nat. Cell Biol. 1, E25-E27
36. de Rooij, J., and Bos, J. L. (1997) Oncogene 14, 623-625
37. Vetter, I. R., Linnemann, T., Wohlgemuth, S., Geyer, M., Kalbitzer, H. R., Herrmann, C., and Wittinghofer, A. (1999) FEBS Lett. 451, 175-180
38. Mosteller, R. D., Han, J., and Broek, D. (1994) Mol. Cell. Biol. 14, 1104-1112
39. Matsubara, K., Kishida, S., Matsuura, Y., Kitayama, H., Noda, M., and Kikuchi, A. (1999) Oncogene 18, 1303-1312
40. Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Oncogene 17, 1415-1438
41. de Rooij, J., Boenink, N. M., van Triest, M., Cool, R. H., Wittinghofer, A., and Bos, J. L. (1999) J. Biol. Chem. 274, 38125-38130
42. Liao, Y., Kariya, K., Hu, C. D., Shibatohge, M., Goshima, M., Okada, T., Watari, Y., Gao, X., Jin, T. G., Yamawaki-Kataoka, Y., and Kataoka, T. (1999) J. Biol. Chem. 274, 37815-37820
43. Ichiba, T., Hoshi, Y., Eto, Y., Tajima, N., and Kuraishi, Y. (1999) FEBS Lett. 457, 85-89
44. Ohtsuka, T., Hata, Y., Ide, N., Yasuda, T., Inoue, E., Inoue, T., Mizoguchi, A., and Takai, Y. (1999) Biochem. Biophys. Res. Commun. 265, 38-44
45. de Rooij, J., Rehmann, H., van Triest, M., Cool, R. H., Wittinghofer, A., and Bos, J. L. (2000) J. Biol. Chem. 275, 20829-20836
46. Ohba, Y., Mochizuki, N., Yamashita, S., Chan, A. M., Schrader, J. W., Hattori, S., Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, 20020-20026
47. Pham, N., Cheglakov, I., Koch, C. A., de Hoog, C. L., Moran, M. F., and Rotin, D. (2000) Curr. Biol. 10, 555-558
48. Iouzalen, N., Camonis, J., and Moreau, J. (1998) Biochem. Biophys. Res. Commun. 250, 359-363
49. Matsuda, M., and Kurata, T. (1996) Cell. Signal. 8, 335-340
50. Dodelet, V. C., Pazzagli, C., Zisch, A. H., Hauser, C. A., and Pasquale, E. B. (1999) J. Biol. Chem. 274, 31941-31946
51. Lu, Y., Brush, J., and Stewart, T. A. (1999) J. Biol. Chem. 274, 10047-10052
52. Posern, G., Weber, C. K., Rapp, U. R., and Feller, S. M. (1998) J. Biol. Chem. 273, 24297-24300
53. Boussiotis, V. A., Freeman, G. J., Berezovskaya, A., Barber, D. L., and Nadler, L. M. (1997) Science 278, 124-128
54. Wojcik, J., Girault, J. A., Labesse, G., Chomilier, J., Mornon, J. P., and Callebaut, I. (1999) Biochem. Biophys. Res. Commun. 259, 113-120
55. Ponting, C. P. (1999) J. Mol. Med. 77, 695-698
56. Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5458-5463
57. Shah, G. V., Rayford, W., Noble, M. J., Austenfeld, M., Weigel, J., Vamos, S., and Mebust, W. K. (1994) Endocrinology 134, 596-602

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
G. P. Katsoulotos, M. Qi, J. C. Qi, K. Tanaka, W. E. Hughes, T. J. Molloy, R. Adachi, R. L. Stevens, and S. A. Krilis
The Diacylglycerol-dependent Translocation of Ras Guanine Nucleotide-releasing Protein 4 inside a Human Mast Cell Line Results in Substantial Phenotypic Changes, Including Expression of Interleukin 13 Receptor {alpha}2
J. Biol. Chem., January 18, 2008; 283(3): 1610 - 1621.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. W. Tew, E. L. Lorimer, T. J. Berg, H. Zhi, R. Li, and C. L. Williams
SmgGDS Regulates Cell Proliferation, Migration, and NF-{kappa}B Transcriptional Activity in Non-small Cell Lung Carcinoma
J. Biol. Chem., January 11, 2008; 283(2): 963 - 976.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Li, J. Yan, P. De, H.-C. Chang, A. Yamauchi, K. W. Christopherson II, N. C. Paranavitana, X. Peng, C. Kim, V. Munugulavadla, et al.
Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins
J. Immunol., December 15, 2007; 179(12): 8322 - 8331.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Tsuboi and J. Meerloo
Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages
J. Biol. Chem., November 23, 2007; 282(47): 34194 - 34203.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
Y. Yoshikawa, T. Satoh, T. Tamura, P. Wei, S. E. Bilasy, H. Edamatsu, A. Aiba, K. Katagiri, T. Kinashi, K. Nakao, et al.
The M-Ras-RA-GEF-2-Rap1 Pathway Mediates Tumor Necrosis Factor-{alpha} dependent Regulation of Integrin Activation in Splenocytes
Mol. Biol. Cell, August 1, 2007; 18(8): 2949 - 2959.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Hong, R. C. Doebele, M. W. Lingen, L. A. Quilliam, W.-J. Tang, and M. R. Rosner
Anthrax Edema Toxin Inhibits Endothelial Cell Chemotaxis via Epac and Rap1
J. Biol. Chem., July 6, 2007; 282(27): 19781 - 19787.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
T. J. Jeon, D.-J. Lee, S. Merlot, G. Weeks, and R. A. Firtel
Rap1 controls cell adhesion and cell motility through the regulation of myosin II
J. Cell Biol., March 26, 2007; 176(7): 1021 - 1033.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Tsuboi
Requirement for a Complex of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein in Podosome Formation in Macrophages
J. Immunol., March 1, 2007; 178(5): 2987 - 2995.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. M. Okamura, C. E. Oki-Idouchi, and P. S. Lorenzo
The Exchange Factor and Diacylglycerol Receptor RasGRP3 Interacts with Dynein Light Chain 1 through Its C-terminal Domain
J. Biol. Chem., November 24, 2006; 281(47): 36132 - 36139.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
S. Huelsmann, C. Hepper, D. Marchese, C. Knoll, and R. Reuter
The PDZ-GEF Dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo
Development, August 1, 2006; 133(15): 2915 - 2924.
[Abstract] [Full Text] [PDF]