INTRODUCTION

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The Ras family of small GTPases (5) is comprised of the classical Ras GTPases (H-Ras, N-Ras, and K-Ras, henceforth collectively referred to as Ras) as well as a more divergent group of Ras-related proteins (TC21, R-Ras, R-Ras3, and the Rals and Raps). By oscillating between GDP-bound forms, which are inert, and GTP-bound forms, which are able to bind and activate multiple effector proteins, these GTPases can serve as switches for the propagation and divergence of signalling pathways. The critical role of Ras in communicating signals from cell surface receptors to intracellular kinases has now been established. Ras is rapidly converted to its GTP-bound form in response to the ligation of many different types of signalling receptors on the cell surface (18). Once GTP bound, Ras can bind to the Raf kinase, bringing it up to the membrane where it can be activated by tyrosine and/or serine phosphorylation by kinases in membrane-localized signalling complexes (46). Raf then initiates a kinase cascade through MEK, ERK1, and ERK2 (56), leading to the phosphorylation and activation of transcription factors such as Elk-1 (61). GTP-bound Ras also binds to and activates phosphatidylinositol 3-kinase, which may lead to the activation of GTPases of the Rho family (57), and binds to and activates RalGDS, thus potentially leading to the activation of Ral GTPases (24). The Ras-related proteins TC21, R-Ras, R-Ras3, and Ral have been relatively neglected, but the available evidence implicates them as collaborators of Ras in signal transduction, and, as suggested for Ral, they may be downstream effectors of Ras which serve to propagate and diversify signalling via GTPase cascades (5, 24, 29). The Rap GTPases are distinct in that they seem to act as repressors of Ras function, possibly by competing with Ras for Raf binding (32).

Like other small GTPases, the Ras family members are themselves controlled by proteins which modulate their nucleotide binding state or their intrinsic GTPase activity. Ras activation appears to be determined largely by specific guanine nucleotide exchange factors (GEFs), which promote the release of GDP from Ras and thus facilitate their conversion to the GTP-bound state (4, 23, 55). A common mechanism of activation of these GEFs is via their translocation to membranes, which is assumed to work simply by bringing the GEF into contact with membrane-bound GTPase targets. Several mammalian GEFs capable of activating Ras or Ras-related GTPases have been identified so far. All of these GEFs have a common GEF domain, which houses their guanine nucleotide exchange activities. Sos1 and Sos2 are a pair of very closely related GEFs which act only on Ras (11). The Sos GEFs are drawn to the membrane in response to tyrosine kinase activation by their interaction with adapter proteins, e.g., Grb2, which binds to phosphorylated tyrosines via its SH2 domain and binds to a proline cluster on Sos via its SH3 domains (18). RasGRF-1 and RasGRF-2 comprise a second class of GEFs which are related to Sos both within the GEF domain and in the possession of PH and DH domains (9, 21, 59). Like the two forms of Sos, both RasGRFs act on Ras. In addition, RasGRF-1 can serve as a GEF for R-Ras (28). The RasGRFs appear to be specialized for activating Ras in response to calcium signalling, via their calmodulin-binding IQ motifs (21, 22). RGL, RLF, and RalGDS are a third group of GEFs, which are homologous to Sos in the GEF catalytic domain but are otherwise quite different in sequence. RalGDS is a GEF specific for the Ral GTPases (1) and is activated by binding to Ras, thus directly coupling Ras activation and Ral activation (24). C3G is a GEF for R-Ras and Rap (27, 28). Its similarity to Sos and the other GEFs is also confined to the GEF domain. It forms complexes with the adapter protein Crk and may thereby be activated in response to tyrosine kinase-coupled receptors.

In lymphocytes, the classical Ras GTPases are activated following ligation of the T-cell receptor or B-cell receptor, as well as by receptors for some cytokines and costimulators (18). All lymphocytes probably express Sos, and this GEF has been implicated in Ras activation occurring downstream of T-cell and B-cell receptors (31, 39, 58, 60). However, in at least some circumstances, T-cell receptor signalling that leads to Ras activation does not seem to result in recruitment of Sos to receptor complexes (8, 49). RasGRF-1 is not expressed by lymphocytes. The expression of RasGRF-2 is much more widespread (21), although it has not yet been determined if this GEF is expressed by lymphocytes. Thus, it is not clear whether the known GEFs are able to mediate all Ras activation events in lymphocytes or whether additional Ras-specific GEFs are required.

In a general attempt to identify additional participants in signalling pathways leading to cell proliferation, we have screened cDNA libraries for clones whose expression causes loss of contact inhibition and morphological transformation of fibroblast cell lines (63). Most of the clones isolated in these screens have been known or novel upstream activators or downstream effectors of Ras or Rho family GTPases. This paper describes the selection from a murine T-cell line of a strongly transforming cDNA which encodes RasGRP, a Ras-specific GEF recently identified via a similar screen for transforming cDNAs derived from rat brain (19). We show that the ability of murine RasGRP (mRasGRP) to transform fibroblasts and activate mitogen-activated protein kinases (MAP kinases) is dependent on its GEF and REM domains, with the C1 domain playing an essential role in mediating the translocation of mRasGRP to cell membranes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cell lines. T28 cells (53) and BOSC 23 retroviral packaging cells (52) (derived from the human epithelial cell line 293T) were cultured in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal calf serum. NIH 3T3 cells were obtained from the American Type Culture Collection and cultured at low density in DME containing 9% calf serum.

cDNA library synthesis, viral transmission, and screening. The vectors and methodology for the construction of cDNA libraries, their conversion to retroviral form, infection of NIH 3T3 cells, selection of transformed cell clones, recovery of proviral cDNAs, and secondary screening for transforming cDNA clones have been described previously (63). One important modification used in the cloning of the CXR-CT cDNA was the use of BOSC 23 packaging cells for production of retroviral libraries, as these cells produce much higher library titers than do the NIH 3T3-derived packaging lines that had been used previously. Transfection of BOSC 23 cells was via calcium phosphate precipitates, as described previously (52). Details relevant to the screening of the T28 library are given below.

Cloning of 5'- and 3'-extended cDNAs and sequence analysis and comparison. cDNAs which overlapped with the original CXR-CT cDNA but extended further 3' or 5' were isolated from the T28 cell cDNA library by PCR amplification with a CXR-specific primer plus vector-specific primers.

Construction of mutant forms of mRasGRP cDNAs. All deletion mutants were made by fusing natural or PCR-generated restriction enzyme sites to start codons, stop codons, green fluorescence protein (GFP) (derived from pEGFP-C1; Clontech), and/or hemagglutinin (HA) epitope tags, provided by pCTV derivative vectors.

Construction of K-Ras mutants. The starting cDNA was an activated human K-Ras clone. This was fused at its N terminus to GFP, making a cDNA that was as fully transforming as was the unmodified K-Ras. The prenylation site was mutated by replacing sequence downstream of a BstBI site with a synthetic oligonucleotide and then ligating into an HA-tagging vector, resulting in a cDNA encoding K-Ras with the C-terminal sequence RKHKEKMSKDGs(HA tag)stop, where the uppercase letters indicate natural K-Ras sequence. The C1 domain of RasGRP was then attached to this modified K-Ras, resulting in the C-terminal sequence RKHKEKMSKDGs(C1 domain)(HA tag)stop. All three forms of K-Ras were expressed at high levels, as determined by flow cytometry.

Detection of expression of HA-tagged or GFP-tagged mRasGRP or K-Ras constructs. Tagged, mutated, and other variants of mRasGRP and control cDNAs in CTV retroviral vectors were converted to retroviral form by transfection into BOSC 23 cells, and then viral supernatants were used to infect NIH 3T3 cells. All mRasGRP constructs were tested for protein expression and structural integrity by Western blot analysis of infected NIH 3T3 cells, using anti-HA monoclonal antibody HA.11 (BABCo) or anti-GFP (Clontech) as a primary antibody and peroxidase-conjugated polyclonal anti-mouse immunoglobulin G (Jackson Laboratories) as a secondary antibody, and detection by chemiluminescence with the ECL system (Amersham). The range of expression in cell populations was determined for GFP-tagged constructs with a FACScan flow cytometer (Becton Dickinson).

Fluorescence analysis of mRasGRP localization. NIH 3T3 cells infected with retroviral vectors expressing GFP-tagged forms of mRasGRP were treated as indicated in the figure legends. The cells were then fixed in 4% paraformaldehyde. For colocalization of GFP-tagged forms of mRasGRP and the endoplasmic reticulum-specific protein BiP, the cells were permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin, and incubated with the anti-BiP monoclonal antibody SPA-827 (StressGen Biotechnologies, Inc., Victoria, British Columbia, Canada), and then with Texas red-conjugated secondary antibody. Photomicrographs were taken under UV illumination, using a Zeiss fluorescent microscope.

Quantification of transformation efficiency. NIH 3T3 cells expressing GFP-tagged derivatives of mRasGRP were plated at very low densities to allow individual colonies to form and were maintained in high-serum medium for 13 days. At that time, colonies were scored as transformed if they showed an obvious absence of contact inhibition and high refractility. The proportion of transformed colonies was normalized for the proportion expressing mRasGRP, as previously determined by flow cytometry. For mRasGRP derivatives which caused transformation, the number of colonies scored was relatively low (20 to 40) because they had to be completely separated to be distinguishable. For derivatives with very low or nil transformation efficiencies, up to 1,000 colonies could be scored, because rare transformed colonies (or their complete absence) could be individually scored despite being surrounded by and merged with a high density of nontransformed colonies (which were countable by extrapolation to very-low-density plates in a serial dilution).

Elk-1 activation assays. The indicated cDNAs were inserted into the pAX142 (15) or pZIPNeo (10) expression vector and cotransfected into NIH 3T3 cells, along with pGal4-Elk-1, which expresses a fusion of the Gal4 DNA-binding domain and the transactivation domain and MAP kinase sites of Elk-1, and with pGal4-LUC, which contains a luciferase reporter driven by a minimal promoter and tandem Gal-4-binding sites (29). The cells were switched to DME containing 0.5% fetal bovine serum at 34 h posttransfection, and 14 h later the cells were lysed and assayed for luciferase activity as described previously (29).

ERK1 and ERK2 activation assays. NIH 3T3 cells stably transduced with the indicated retroviral vectors were maintained in DME containing 9% calf serum or switched to serum-free DME for 2 h and then treated as indicated. BOSC 23 cells were transiently transfected with the indicated cDNAs in pAX142, using the calcium phosphate procedure (52). At 32 h after transfection, the BOSC 23 cells were switched to serum-free RPMI medium containing 4 µg of insulin per ml and 5 ng of sodium selenite per ml for 19 h and then were stimulated for 15 min with 10% fetal calf serum as indicated. Total cell lysates were prepared and analyzed for activation of ERK1 and ERK2 by Western blotting with a polyclonal antibody specific to ERK1 and ERK2 phosphorylated at Tyr204, using the procedures specified by New England Biolabs.

Hybridization analysis of RNA. Total cellular RNA was separated on 5% formaldehyde agarose gels and transferred to a Hybond N+ nylon membrane (Amersham). Hybridization and high-stringency washing were performed as described previously (20). The probe was a 1,250-bp fragment of the CXR-CT cDNA which encompasses the region encoding amino acids 49 to 448.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Selection of a cDNA clone which causes Ras-like transformation of NIH 3T3 cells. To identify novel Ras activators expressed by T lymphocytes, we screened for cDNAs capable of transforming NIH 3T3 cells within a large cDNA library made from a murine T-cell hybridoma, T28. This cell line has functional signalling from CD3 and can also be activated by cotreatment with phorbol ester and calcium ionophores (36a). The cDNA library was made in plasmid form in the pCTV1 retroviral vector, converted to retroviral form by transfection into the packaging cell line BOSC 23, and then transferred to NIH 3T3 cells by infection. Among the approximately 25 transformed cell foci arising in the infected NIH 3T3 cultures was one which had the refractile, swirling morphology characteristic of Ras-induced transformation. This cell clone was picked and expanded, and the single proviral cDNA within it was recovered by PCR and recloned into the pCTV3 retroviral vector to produce clone pCXR-CT. Infection of NIH 3T3 cells with retrovirus derived from pCXR-CT resulted in massive transformation of the cell culture (Fig. 1), with essentially every infected cell in the culture becoming refractile. The cell cultures continued to proliferate beyond confluence, eventually peeling off the culture surface in large masses. The onset of obvious morphological transformation occurred about 2 days later than that induced by expression of activated mutants of N-Ras or K-Ras.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   Transforming activity of the CXR-CT cDNA clone encoding truncated mRasGRP. NIH 3T3 cells were plated at low density and infected with CTV83B, which is an empty retroviral vector (Control); with retroviral vector CTV82, carrying the CXR-CT cDNA (CXR); or with retroviral vector CTV80, carrying a cDNA encoding N-Ras activated by a Q61K mutation (N-Ras). After 8 days (5 days postconfluence), the cell cultures were stained with methylene blue.

View larger version (53K): [in this window] [in a new window]   FIG. 2.   (A) Sequence of the mRasGRP peptide. The peptide sequence is derived from the continual open reading frame obtained from the combined sequences of the CXR-CT cDNA and four overlapping cDNAs isolated from the T28 cell library. The REM and GEF domains are indicated by boldface, while the EF hands and C1 domain are indicated by the solid and dotted underline, respectively. Potential MAP kinase phosphorylation sites (16) are underlined. The potential amphipathic -helix is double underlined, with bars under the aliphatic residues at seventh positions in the sequence. The arrows indicate the boundaries of the peptides encoded by the CT, FL, and XFL forms of mRasGRP and the various C-terminal deletions. The open and closed circles indicate positions affected by point mutations in CXR-GEFµ and CXR-Proµ, respectively. (B) Comparison of C1 domain sequences. The C1 domain of mRasGRP is aligned with C1 domains that bind phorbol ester (the second C1 domains of PKC-, PKC-, and PKC- and the single C1 domain of n-chimerin) and those that do not bind phorbol ester (the single C1 domains of PKC-, Raf, and Vav). Columns of histidines and cysteines involved in zinc binding are marked with asterisks. Residues predicted to be capable of participating in phorbol ester binding have solid highlighting.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Mutational analysis of regions of mRasGRP required for transformation of NIH 3T3 cells. The domain structure of mRasGRP is illustrated in the upper diagram (REM, Ras exchanger motif; GEF, region of homology to the GEF domain of Sos; P, proline cluster; EFH, each box represents one EF hand motif; C1, C1 domain; , putative -helical segment). Deletion and other mutant constructs are depicted in the other diagrams. All constructs are GFP tagged at N termini and/or HA tagged at C termini (or between the mRasGRP sequence and the prenylation signal). The prenylation signal is symbolized by the black circle, and sites of point mutations are symbolized by the black triangles. Exact boundaries of domains and predicted translation products of the deletion and mutant constructs are indicated in Fig. 2, as are positions of point mutations. The open box in construct C1/PKC represents the second C1 domain of PKC-. The stippled box represents the region of K-Ras N terminal to its prenylation signal. Transformation efficiency in high-serum medium is the proportion of isolated colonies expressing N-terminally GFP-tagged, retrovirally transduced mRasGRP constructs which were morphologically transformed and not contact inhibited after 13 days in culture in medium with 9% calf serum. The spontaneous rate of transformation was less than 1 focus/105 cells. Transformation in low-serum medium was assessed after 6 days of culture with daily feedings of medium containing 0.5% fetal bovine serum and 4 µg of insulin per ml, with or without 10 ng of PMA per ml.

The GEF domain of mRasGRP is essential for its transforming activity. Amino acids 201 to 371 of mRasGRP are clearly homologous to the GEF domains of Sos and related exchange factors which are known or assumed to act on one or more members of the Ras family of GTPases (4, 23, 55). Among mammalian GEFs, this region of RasGRP is most similar to RasGRF and C3G and somewhat less similar to Rgl, RalGDS, and Sos1 (determined by using pairwise similarity scores derived by the Genetics Computer Group Pileup program). Similarities to all GEFs are particularly strong within the conserved SCR-1, -2, and -3 boxes within the GEF domain (4). The first residue in the SCR-2 box is invariably arginine in all Sos family members, and mutation of this residue to glutamate in the Saccharomyces cerevisiae CDC25 GEF eliminates its function, apparently by preventing interaction with Ras while otherwise retaining the structure and catalytic competence of the GEF domain (51). An equivalent arginine-to-glutamate mutation at position 271 in mRasGRP eliminates its ability to transform NIH 3T3 cells (Fig. 3, GEFµ). The expression of GEFµ was somewhat lower on average than the expression of CXR-CT (Fig. 4). However, about 10% of the GEFµ cells (none of which were transformed) expressed amounts of mRasGRP protein equivalent to or greater than the levels expressed by 50% of the CXR-CT cells (of which at least 80% were transformed). Therefore, in cells expressing equivalent levels of protein, the point mutation in the GEF domain eliminated the ability of mRasGRP to induce transformation.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Expression levels of mRasGRP in retrovirally transduced NIH 3T3 cells. mRasGRP constructs were tagged by fusion of GFP to the CXR-CT N terminus. Expression levels were determined by flow cytometry just prior to plating of cells for the transformation assays shown in Fig. 3. The histograms show the distribution of fluorescence values in the population of cells expressing the indicated GFP-tagged mRasGRP construct, after gating out of cells with fluorescence values not above those of control, non-GFP-expressing cells. Fluorescence intensity is on a log scale.

A C1 domain within mRasGRP is required for its transforming activity. A unique feature of the C-terminal region of mRasGRP is the presence of a C1 domain (33) very similar to those found in classical and novel protein kinase Cs (PKCs). In most of these PKCs (and in some other proteins, such as n-chimerin), the C1 domains serve as binding sites for diacylglycerol or phorbol ester and act as positive regulatory domains by mediating the translocation of the C1 domain-containing protein to membranes enriched in diacylglycerol or phorbol ester (47, 48). In addition to the histidines and cysteines required for zinc coordination, the C1 domain of mRasGRP contains appropriate residues at the 11 positions that participate in forming a phorbol ester-binding pocket in the second C1 domain of PKC- (64) (Fig. 2B). C1 domains which do not bind phorbol ester, e.g., those in Raf, Vav, or the atypical PKC-, have inappropriate residues or gaps in at least three of these positions. Thus, the C1 domain of mRasGRP is predicted to bind phorbol ester and diacylglycerol and by implication is expected to regulate the function of mRasGRP by mediating its binding to membranes enriched in the second messenger diacylglycerol.

EF hands and a potential SH3 domain-binding site are not needed for transformation via mRasGRP. The region between the N-terminal GEF domain and the C-terminal C1 domain contains two features which could potentially contribute to the regulation of mRasGRP. The first is a pair of EF hands (Fig. 2), compact domains which serve as sensors of calcium concentration changes in the cell (34). EF hands typically function in pairs, making cooperative changes in conformation following calcium binding which create hydrophobic surfaces that can participate in intra- or interprotein interactions. The EF hand pair of mRasGRP thus has the potential to serve as a calcium-responsive element that could alter interdomain interactions within mRasGRP or alter interactions of mRasGRP with other proteins or with membranes. However, deletion of the EF hand pair (Fig. 3, EF) or point mutations in the putative calcium-binding residues of the second EF hand (data not shown) had no effect on transformation activity. The second feature is a proline cluster (Fig. 2) which could serve as a binding site for SH3 domains (RapPLtPsKppvvv, where the uppercase letters indicate residues that could participate in SH3 binding [44]). A cluster of point mutations which eliminate the potential for SH3 binding (Fig. 3, Proµ) in this sequence had no effect on transformation activity. Double mutants of mRasGRP lacking both the EF hand pair and the proline cluster were also fully active in transformation assays (data not shown). Therefore, the EF hands and the proline cluster, either singly or collectively, have no discernible influence on the function of mRasGRP in the transformation assay.

Localization of mRasGRP to cell membranes in response to either serum or phorbol ester. In serum-starved NIH 3T3 cells, the CXR-CT form of mRasGRP is distributed relatively uniformly throughout the cell, including the nucleus (Fig. 5). After a 15-min stimulation with serum or the phorbol ester phorbol 12-myristate 13-acetate (PMA), CXR-CT became predominantly localized to cellular structures concentrated around the nucleus and in a punctate network spreading further out into the cell periphery (Fig. 5). Translocation of CXR-CT was induced by PMA at concentrations as low as 0.5 ng/ml. Most of the mRasGRP in serum- or PMA-stimulated cells was in the endoplasmic reticulum, as determined by its colocalization with the endoplasmic reticulum-specific protein BiP (data not shown). The occurrence of mRasGRP in BiP-negative structures around and to one side of the nucleus probably represents localization to the nuclear envelope and Golgi. An equivalent concentration of some PKC isoforms in the endoplasmic reticulum and Golgi is seen in PMA-stimulated fibroblasts (26).

View larger version (105K): [in this window] [in a new window]   FIG. 5.   Translocation of mRasGRP in response to serum or PMA stimulation. NIH 3T3 cells stably expressing the indicated forms of N-terminally GFP-tagged mRasGRP via retroviral infection were serum starved in DME for 4 h. The medium was then replaced with DME (Nil), DME containing 10% calf serum (serum), or DME containing 50 ng of PMA per ml (PMA). The cells were fixed 15 min later, UV illuminated, and photographed.

View larger version (92K): [in this window] [in a new window]   FIG. 6.   Translocation of C1 domains in response to serum or PMA stimulation. NIH 3T3 cells were infected with retroviral vectors expressing GFP alone (GFP) or fusions of GFP to the C termini of isolated C1 domains of mRasGRP (GFP/CXR-C1) or PKC- (GFP/PKC-C1). Cells were serum stimulated, serum deprived for 3.5 hours (Nil), or serum deprived and then stimulated with 50 ng of PMA per ml for 1 or 15 min. The cells were then fixed and photographed under UV illumination.

View larger version (95K): [in this window] [in a new window]   FIG. 7.   Phospholipase C-induced localization of mRasGRP and isolated C1 domains. NIH 3T3 cells infected with retroviral vectors expressing GFP alone, fusions of GFP to the isolated C1 domains of mRasGRP (GFP-CXR-C1) or PKC- (GFP/PKC-C1), or GFP fusions of the indicated forms of mRasGRP were serum deprived for 3.5 h in DME, and then Bacillus cereus PC-PLC (Sigma) was added to the medium at 4 U/ml and the cells were cultured at 37°C for 45 min. The cells were then fixed and photographed under UV illumination.

mRasGRP expression induces MAP kinase activation. The transcription factor Elk-1 is activated by C-terminal phosphorylation via the MAP kinases ERK-1 and ERK-2, as well as by the related MAP kinase JNK (61). These MAP kinases are themselves indirectly activated in fibroblasts by GTP-loaded Ras, in the case of the ERKs via a kinase cascade from Raf to MEK kinases (18). We have used an assay based on MAP kinase-dependent stimulation of transactivation of a reporter gene via the C-terminal domain of Elk-1 (29) to indirectly measure mRasGRP activity and to determine the ability of mRasGRP to act in conjunction with specific Ras family members. Expression of mRasGRP caused a very high level of MAP kinase activation in NIH 3T3 cells cultured in low-serum medium, equivalent to that attained by expression of a prenylated and thus hyperactivated form of RasGRF-1 (Fig. 8A). A prenylated form of mRasGRP (CXR-C3/pr) induced fourfold-higher levels of MAP kinase activation, while the point mutation in the GEF domain and the deletion of the C1 domain eliminated the ability of mRasGRP to activate MAP kinases (Fig. 8A). MAP kinase activation by mRasGRP was increased by coexpression with normal forms of either H-Ras, K-Ras, or N-Ras but not by coexpression with the Ras-related GTPase R-Ras or TC21 (Fig. 8A). Expression of these GTPases on their own had only very minor effects on MAP kinase activation (data not shown). Coexpression of dominant negative forms of H-Ras and K-Ras (which bind to GEFs but are not substrates for guanine nucleotide exchange) partially suppressed MAP kinase activation via mRasGRP, while dominant negative R-Ras had no effect (Fig. 8A). The dominant negative forms of H-Ras and K-Ras did not inhibit MAP kinase activation via expression of an activated form of Raf-1 (data not shown), indicating that they were acting specifically on the process of Ras activation rather than at a later point in the transduction of activating signals from mRasGRP to MAP kinases. mRasGRP also strongly induced expression from NF-B-, Ets/AP1-, and cyclin D-responsive promoters, each of which is inducible via Ras activation (data not shown). For all three of these Ras-responsive reporter systems, mRasGRP stimulated expression equivalently to prenylated RasGRF, while prenylated mRasGRP was considerably more effective.

View larger version (26K): [in this window] [in a new window]   FIG. 8.   Induction of MAP kinase activation and ERK1 and -2 phosphorylation by expression of mRasGRP. (A) NIH 3T3 cells were cotransfected with Gal4-LUC and Gal4-Elk-1, along with expression vectors expressing mRasGRP cDNAs, prenylated RasGRF-1, and normal or dominant negative forms of Ras family GTPases. The data shown are representative of those from three separate experiments, with data for each point determined in triplicate in each experiment. (B) NIH 3T3 cells were stably expressing the indicated mRasGRP cDNAs by retroviral infection. BOSC 23 cells were transiently transfected with the indicated cDNAs. Cells were stimulated with serum as described in Materials and Methods and then analyzed for ERK1 and ERK2 phosphorylation by Western blotting. Equivalent loading of protein was checked by Coomassie blue staining and by reprobing the blot with an anti-ERK1 and -2 antibody that was not phosphorylation specific.

The C1 domain of mRasGRP mediates PMA-dependent transformation in low concentrations of serum. When the amount of serum in the culture medium was reduced from the normal concentration of 9% down to 0.5%, NIH 3T3 cells expressing CXR-FL or CXR-CT lost their transformed appearance and stopped proliferating. This reversion of the transformed phenotype allowed us to determine if PMA could regulate the ability of mRasGRP to induce cell transformation. Addition of PMA to the low-serum medium caused CXR-CT- and CXR-FL-expressing cells to regain their transformed appearance within 1 day and also enabled them to proliferate beyond confluence (Fig. 3). Control cells grew to higher density in the low-serum medium when it was supplemented with 10 ng of PMA per ml, but they were not morphologically transformed and were fully contact inhibited. Morphological transformation of mRasGRP-expressing cells in low-serum medium was induced by PMA concentrations as low as 0.1 ng/ml. PMA-dependent transformation in low-serum medium was also observed with cells expressing the Proµ or EF form of mRasGRP (Fig. 3). The C1 form lacking the C1 domain as well as the more extensive CXR-C3 C-terminal deletion and the REM and GEFµ mutants did not cause morphological transformation in the presence of PMA concentrations as high as 300 ng/ml. In contrast, cells expressing the prenylated form of mRasGRP (Fig. 3, C3/pr) were transformed in low-serum medium in the presence or absence of PMA, as were cells expressing GTPase-defective K-Ras (Fig. 3). These results demonstrate that PMA can regulate the ability of mRasGRP to induce transformation when the serum concentration is low. This effect of PMA requires the C1 domain of mRasGRP but not its EF hands or proline cluster, and the requirement for PMA or the C1 domain is abrogated by constitutive plasma membrane localization of mRasGRP.

mRasGRP can induce transformation via a C1 domain-independent mechanism. None of the cells expressing the C1 form of mRasGRP were detectably transformed when the cells were continually passaged below confluence or in cultures that had been maintained as monolayers for up to 10 days. Beyond that time, highly transformed cells began to gradually appear, until after 28 days they formed the majority of cells in the culture. This accumulation represented both overgrowth of the monolayer by transformed cells and the transformation of previously nontransformed cells. Conversion of the majority of cells in an isolated colony to a highly transformed phenotype could occur over a few days. This conversion presumably reflects de novo activation of mRasGRP, because there is no enhanced activation of MAP kinases in the C1-expressing cells prior to their conversion to a transformed phenotype (Fig. 8A). Amounts of C1 protein were equivalent among both nontransformed and transformed cells (data not shown), indicating that transformation did not result from unusually high levels of CXR-C1 expression in the subset of cells that became transformed or that transformation was caused by a shift to higher levels of expression of C1.

Expression pattern of mRasGRP. Northern analysis of total RNA identified a predominant mRasGRP transcript of about 4.8 kb in T28 cells (Fig. 9). The same mRNA species was abundant in thymus, at moderate levels in brain, and at lower levels in spleen and bone marrow. No RasGRP transcripts were detectable in kidney, lung, stomach, and skeletal muscle (Fig. 9) or in heart or testis (data not shown). In addition to the T28 T-cell hybridoma, the RasGRP mRNA was detected in the MBL-2 T-cell line; the B-cell lines WEHI 231, ABE8, and A20; the primitive hemopoietic line B6SutA; and P388D₁, a cell line which was derived from a B lymphoma but subsequently underwent monocytic differentiation (Fig. 9). RasGRP transcripts were not detected in the R1.1 or YAC-1 T-cell line, the NSF-70 or Ba/F3 B-cell line, the 32D or DA-3 myeloid cell line, or the GM979 erythroid cell line (Fig. 9). RasGRP transcripts were also not detectable in the murine fibroblast cell line NIH 3T3 or C3H10T1/2 or in the breast-derived cell line C127 (data not shown). Overall, this suggests that RasGRP is expressed in some but not all murine lymphoid cell types and possibly also in uncommitted hemopoietic precursors, as represented by the B6SutA cell line.

View larger version (40K): [in this window] [in a new window]   FIG. 9.   Expression of mRasGRP in murine tissues and hemopoietic cell lines. Northern blots of total RNAs from the indicated tissues of a 6-week-old C57BL/6J mouse or murine hemopoietic cell lines were probed with the CXR-CT cDNA. Marker sizes are indicated.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The GEF function of RasGRP. Purified rat RasGRP catalyzes guanine nucleotide exchange on H-Ras but not on R-Ras (19). Our results obtained by using coexpression of mRasGRP with various Ras family members indicate that this specificity is retained in vivo and that mRasGRP acts on K-Ras and N-Ras as well as H-Ras. The failure of the catalytic domain mutant of mRasGRP to induce transformation suggests that the in vivo function of RasGRP requires direct activation of Ras GTPases via guanine nucleotide exchange. The ability of the REM-plus-GEF region of mRasGRP to induce transformation when prenylated indicates that GEF activity is probably the only function of RasGRP required for transformation, as the only known function of the REM box is to increase the efficiency of GEF catalysis in vitro (37). It remains possible that beyond serving as a Ras-specific GEF, RasGRP has an additional biological function that is not apparent via fibroblast transformation assays.

Regulation of membrane localization of RasGRP by serum, PMA, and diacylglycerol. RasGRP is found predominantly in internal cell membranes in serum-stimulated fibroblasts. This is dependent on its C1 domain, as demonstrated by the equivalent localization pattern of the isolated C1 domain of mRasGRP and the absence of membrane localization of the mRasGRP mutant lacking the C1 domain. The C1 domain of RasGRP is very similar in sequence to the C1 domains of PKCs and n-chimerin, which bind phorbol esters and diacylglycerol. This suggests that membrane localization of RasGRP could be driven by binding of its C1 domain to phorbol ester or diacylglycerol. The best evidence for this mechanism of membrane localization is the complete equivalence of the C1 domains of mRasGRP and PKC- in their abilities to restore serum- and PMA-dependent transformation competence to mRasGRP deletion mutants, their localization in serum-stimulated cells, their transient translocation to the plasma membrane in response to PMA, and their concentration in lipid droplets following PC-PLC treatment.

Potential roles of RasGRP in lymphocytes. Ras activation can be induced in lymphocytes by tyrosine kinase-coupled receptors for antigen, cytokines, or costimulators, by G protein-coupled receptors, and by phorbol ester treatment (8, 18, 45). Given that most of these trigger or mimic diacylglycerol production, there is considerable potential for RasGRP to participate in the diverse processes that activate Ras in lymphocytes.