Di-Ras, a Distinct Subgroup of Ras Family GTPases with Unique Biochemical Properties

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30–40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family. The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30–40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family. The small GTPase Ras family acts as a molecular switch that regulates a wide range of cell functions including proliferation and differentiation (1Bos J.L. Biochim. Biophys. Acta Rev. Cancer. 1997; 1333: M19-M31Google Scholar, 2Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Google Scholar, 3Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar). Ras is activated in response to a variety of extracellular signals, resulting in stimulation of tyrosine kinases either directly or indirectly. The small GTPase, which cycles between active GTP-bound and inactive GDP-bound states, is activated by guanine nucleotide exchange factors that enhance the exchange of bound GDP for GTP and is deactivated by GTPase-activating proteins that increase the intrinsic rate of hydrolysis of bound GTP. The GTP-bound form of Ras associates with several effector molecules, most notably members of the Raf family, the RalGDS family and phosphoinositide 3-kinase. Amino acid residues 32–40 of Ras are important for its interaction with effector molecules and designated as the “effector domain” (4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Google Scholar). The biological consequences of their interactions depend greatly on the cell type and on the context of other signaling events. The members of Ras family, at least 13 at present, are characterized by extensive similarities in their effector domains (5Bos J.L. EMBO J. 1998; 17: 6776-6782Google Scholar). Besides three Ras proteins (Ha-Ras, Ki-Ras, and N-Ras), there are four Rap proteins (Rap1A, Rap1B, Rap2A, and Rap2B), two Ral proteins (RalA and RalB), R-Ras, TC21, R-Ras3/M-Ras, and Rheb in the Ras family. Although many Ras members can interact with the same effector molecules as the three Ras proteins, the physiological roles of most Ras-like GTPases are not fully understood. More recently, some members of the Ras family have begun to be analyzed. For example, Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it functions independently or coordinately with Ras-mediated signaling (6Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Google Scholar). Rap1 was originally discovered as a suppressor of Ki-Ras-induced transformation, and it has been considered to function as an antagonist of Ras signaling by trapping Ras effectors, particularly the Ser/Thr kinase Raf (7Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Google Scholar). However, recent studies show that Rap1 is activated by extracellular signals through several regulatory proteins, and raise the possibility that it may function in diverse biological processes, ranging from modulation of cell growth and differentiation to secretion, integrin-mediated cell adhesion, and morphogenesis. It is thus expected that further studies on other members of Ras-like GTPases would also reveal novel signaling pathways involved in a variety of cell functions. In the present study, we report two novel members, Di-Ras1 and Di-Ras2, belonging to a distinct branch of the Ras GTPase family. Although clearly related in sequence, Di-Ras proteins possess different biochemical and functional properties from other members of the Ras family. It is expected that Di-Ras may be involved in novel cellular functions distinct from other Ras-related GTPases. Data base searches using Entrez (NCBIs search and retrieval system) were performed to find novel Ras-related GTPases. Among the many candidate genes in the retrieval results was a cosmid clone (accession numberAC006538) that contains a hypothetical Ras-related gene, which we designated Di-Ras1. Sequence data from the open reading frame of the cosmid were used to design specific primers to amplify the cDNA fragment of Di-Ras1. Primers used wereDi-Ras1F, 5′-ATGCCGGAACAGAGTAACGAT-3′, andDi-Ras1R, 5′-TCACATGAGGGTGCATTTGCC-3′. PCR amplification was performed using human whole brain cDNA (Clontech) and KOD polymerase for 30 cycles (15 s at 98 °C, 2 s at 65 °C, 30 s at 74 °C). The obtained PCR products were sequenced and used to screen a human brain λ ZAPII cDNA library. Of 8.6 × 106 plaques screened, eight positive clones were isolated and sequenced. Four of the eight positive clones were identified as Di-Ras1 cDNA, and the residual four positive clones were overlapping cDNA clones homologues to Di-Ras1 cDNA (thus designatedDi-Ras2). The 5′-cDNA fragments of Di-Ras1were amplified using human brain Marathon Ready cDNA and Advantage cDNA PCR Kits (Clontech) according to the manufacturers instructions. DNA-sequencing reactions were performed using the DYEnamic ET Terminator Cycle Sequencing Premix Kit (AmershamBiosciences). Samples were electrophoresed on an ABI 373 DNA Sequencer and analyzed by ABI Prism model version 2.1.1 software (PE Applied Biosystems). Multiple alignment of Di-Ras proteins to related protein sequences was carried out using CLUSTALW and viewed in GeneDoc. An unrooted phylogenetic tree was prepared using CLUSTALW and viewed in TREEVIEW. Expression patterns ofDi-Ras1 and Di-Ras2 mRNAs were analyzed using human multiple tissue Northern blot (Clontech). Hybridization was carried out in the ExpressHyb hybridization solution (Clontech) in the presence of the [32P]cDNA probe according to the manufacturers instructions. Hybridized blots were washed twice with 2× SSC (1× = 0.15 m NaCl and 0.015 m sodium citrate) and 0.1% SDS, twice with 0.5× SSC and 0.1% SDS, and once with 0.2× SSC and 0.1% SDS at 68 °C. Filters were exposed to x-ray film (Fuji film) at −80 °C for 2−3 days with an intensifying screen. Di-Ras1 and Ha-RascDNAs corresponding to the 1–194 and 1–180 amino acids, respectively, were cloned in pGEX-4T-1 vector to express GST fusion proteins. Overnight culture of Escherichia coliBL21-CodonPlus DE3 (Stratagene) containing the expression vector was diluted 1:50 with a 2× YT medium containing 50 μg/ml ampicillin and grown at 37 °C for ∼2 h (A 600 = ∼0.5), followed by induction with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside at 20 °C for 14 h. The cells (250 ml of culture) were collected by centrifugation and suspended in 20 ml of buffer A consisting of 50 mm Na-Hepes (pH 7.4), 100 mm NaCl, 5 mm MgCl2, 5 mmDTT, 1The abbreviations used are: DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; GTPγS, guanosine 5′-O-(thiotriphosphate); MAPK, mitogen-activated protein kinase; RBD, Ras-binding domain; GST, glutathioneS-transferase; UTR, untranslated region. 2 μg/ml aprotinin, and 0.5 mm Pefabloc SC (Roche Molecular Biochemicals). The suspension was sonicated 10 times for 2 s in ice-cold water and mixed with 0.1 volumes of 10% (w/v) CHAPS. The suspension was rotated at 4 °C for 30 min. After centrifugation (100,000 ×g, 4 °C, 1 h), the clear supernatant was applied to a glutathione-Sepharose 4B column (0.8-ml bed) that had been equilibrated with 5 ml of buffer B consisting of 20 mmNa-Hepes (pH 7.4), 100 mm NaCl, 5 1 mm DTT, and (w/v) CHAPS. After the column with the same proteins were from the column with 5 ml of buffer consisting of 100 mm Na-Hepes (pH 7.4), 5 mm MgCl2, 10 and (w/v) CHAPS. The proteins were applied to a column bed) that had been equilibrated with 30 ml of buffer consisting of 50 mm Na-Hepes (pH 7.4), 20 mm NaCl, 5 mm MgCl2, 1 mm DTT, and (w/v) CHAPS, and the containing GST fusion proteins were collected and at −80 °C to the proteins was performed as proteins were at 30 °C with different of the in a of of buffer consisting of mm Na-Hepes (pH 7.4), 1 mm MgCl2, 1 mm DTT, and (w/v) CHAPS. After for were diluted with of an ice-cold buffer 20 mm MgCl2, and 100 mm and through a The was washed three times with 2 ml of the ice-cold buffer and at °C. on the was by a For nucleotide proteins were with the in a of as A of was and of the were at the times the at 30 °C. were diluted with the buffer and to the as The rate of GTP hydrolysis was performed as proteins were at 30 °C with 1 in a of of buffer B containing of and The was as R. Scholar). After of the of the were and mixed with of ice-cold (w/v) in 50 mm The was at for 5 at 4 °C, and the supernatant was analyzed for the of the and HEK293T cells were in medium with 10% in a of at 37 °C. The cells were with 1 or or of DNA using For kinase the cells were for h and for an in medium containing 0.1% PCR was used to the Di-Ras and sequences into a vector that the at its amino Expression of the proteins was by of or cells followed by with an bound to the proteins were analyzed as T. M. Y. Scholar). the which had been in for h were for 4 h with in The cells × 106 were with 1 ml of an ice-cold buffer mm 100 mm NaCl, 20 mm 1 mm 1 mm DTT, (w/v) 2 μg/ml aprotinin, and 0.5 and and the were with the and protein at 4 °C for 2 h. After extensive of the were by and with a (Fuji A was performed according to the J. H. H. T. J. Biol. Di-Ras2, and corresponding to the and 1–180 amino acids, respectively, were cloned in Ras-binding domains of and corresponding to the and amino acids, respectively, were cloned in was with the and were grown on a medium and were days and for For of ERK, the cells were washed once with an ice-cold and in of a buffer SDS, (w/v) 50 mm and Cell were sonicated and for 5 min. Samples were to and to a the of ERK, was using an that specifically kinase The were using for the of using a according to the manufacturers of Di-Ras1 and cloned into vector (Clontech) for expression of fusion proteins. HEK293T cells were with 1 of DNA using After a cell were prepared and to using an (Clontech). For the cells were grown on at the of a with a and with 1 of DNA as After the cells were viewed with a identified novel members of the Ras GTPase family, Di-Ras1 and Di-Ras for a distinct of family. Di-Ras1 was identified in the data base for Ras-related GTPases as that a cosmid clone from the contains an open reading which a Ras-related the of the gene, PCR was performed using human brain cDNA library. obtained a PCR with the expected molecular of which the nucleotide the the cosmid The resulting PCR was used to screen a human brain λ ZAPII cDNA positive were isolated from 8.6 × 106 and their were sequenced. with that four of the eight were from the Di-Ras1 The nucleotide of the four clones a of 68 a of and of amplification of cDNA identified further a sequence, which a of the predicted The predicted protein was designated as Di-Ras1. The four of the eight were overlapping clones to The nucleotide a of with a of the predicted a of and of The predicted a protein highly related to Di-Ras1 thus we designated it as are some DNA sequences in the data of which sequences are to Di-Ras1 and A amino acid alignment of human Di-Ras and other members of the Ras family. Di-Ras1 amino acid identity with Rap2A, with and with Besides these Ras members, Di-Ras proteins share with a identified suppressor Y. M. Scholar). An unrooted phylogenetic tree of Di-Ras and other members of the Ras family that Di-Ras2, and can be into a distinct of the Ras family 1 several overlapping clones of and sequences are highly to human Di-Ras1 not In search of the protein data base that Di-Ras proteins share with two predicted products of and However, we not find of human Di-Ras in and of and proteins. amino acid sequences from the genes were using Amino acid residues conserved among proteins are in and with are of and are and respectively, in the Entrez data searches of the predicted Di-Ras sequences that they a highly conserved a effector domain to the amino acid 32–40 of and the is is an amino and is amino at the However, Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and positions that are known to be critical for GTP hydrolysis in the Ras family. These positions in Ras, substituted with a variety of amino acids, by Ras for GTP hydrolysis and to the stimulation by proteins, Ras to be as an activated J. M. J. Biol. Scholar, M. Scholar). Moreover, the effector Di-Ras from most Ras members in that Di-Ras1 and and are substituted for Asp-33 and in Di-Ras be in the cDNA that can be into the the expression Northern was performed using a human Northern Di-Ras1 was in brain and was expressed specifically in brain expressed quite low or of of the in human brain that was expressed at a level in the the and the and The expression level was quite low in and The expression of to that of Di-Ras1 that its expression was in the the biochemical properties of we expressed it in as a GST fusion protein and it using a glutathione-Sepharose A the of to the in the presence of 10 mm More of bound to of that of the protein is and the The nucleotide to Di-Ras1 of because the at 100 was of that at 10 mm not The properties of Di-Ras1 were also the of 4 that the for was was by of with GTP and GDP for with at 0.5 and 2 respectively, that the of Di-Ras1 for GTP is that for In not for the at a as as 100 Di-Ras1 to specifically guanine 5 A the of from Di-Ras1 in a with that from and were with and of the bound nucleotide was in the presence of 100 The was from it was on the same that Di-Ras1 the of GTP hydrolysis by and GTP hydrolysis at a low of In GTPase activity of was quite low the of and low A form of Di-Ras1 that the GST the same biochemical properties as of Di-Ras1 not the nucleotide form with Di-Ras1 and Di-Ras2 in living cells. For the proteins were expressed in and and the proteins were by of Expression of the was by not The guanine with the were analyzed by that predominantly as a GDP-bound of was a GTP-bound form in cells. In and of Di-Ras1 and Di-Ras2, respectively, as GTP-bound in the cells. A of the Di-Ras GTP-bound form was also in cells. These results that Di-Ras proteins are predominantly GTP-bound in living cells. has that members of the Ras family their biological through the of several downstream pathways (4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Google Scholar, Curr. Opin. 1997; Scholar). of the pathways activated by Ras is a signaling which the of protein As an to the biological function of we the of Di-Ras to the cells with Ras or Di-Ras were and the cell were The of in these cells was by of the cell using an that specifically kinase. As in of kinase activity in however, Di-Ras1 its activated the of Di-Ras2 also had no on the kinase thus the of Di-Ras to the is with its to Raf. The interaction between Di-Ras1 and Raf was using a a vector containing the cDNA fragment of Di-Ras1 that of to to the As a positive a was with a cDNA that has been to to of the Raf family J. Mol. Cell. Biol. Scholar). of and corresponding to the and amino acids, respectively, were cloned in B that of to was clearly by the stimulation of activity of the In Di-Ras1 Di-Ras2 of Raf. from these results that the of Di-Ras to the is because of its to Raf. also Di-Ras other molecules, as phosphoinositide 3-kinase. However, expression of Di-Ras in cells not which is a downstream of the kinase not It thus that Di-Ras not interact with the Ras-binding domain of phosphoinositide the of Di-Ras proteins and their functions in living HEK293T cells were with containing of Di-Ras at the of of the cells were to with an only a of was that the in the cells the fusion proteins. Interestingly, level expression of or large and the proteins were to the as as the the proteins were with in the cells their expression were low and The large was also Di-Ras proteins were expressed in HEK293T or cells not In expression of a vector or the which the sequence, signals and not cellular at expression and As the presence of the is for and biological functions of Ras family proteins, the of is also to be of Di-Ras proteins. the with of Ha-Ras, because has also been to cellular in some cell T. Y. M. H. M. M. Y. Oncogene. Scholar). However, the by was and different from the at least in HEK293T cells not The expression of also a in the of cells not that the of Di-Ras is not specific to by of the fusion proteins in HEK293T cells. HEK293T cells were grown on and with as After the cells were viewed with a of HEK293T cells and that expression of Di-Ras1 and Di-Ras2 to the of large and corresponding 10 have the and of novel members of the Ras family, human Di-Ras1 and Di-Ras2, which form a distinct branch of the Ras family. The Ras GTPase family exists as an inactive GDP-bound and is to an active GTP-bound in response to a variety of extracellular Di-Ras has a domain that is highly conserved in the GTPase family, and we have that Di-Ras1 GTP. It is that Di-Ras1 GTP and quite low of intrinsic GTPase Di-Ras proteins expressed in and cells were to be in GTP-bound Although we out the possibility that the on Di-Ras in living cells might from of the proteins, the biochemical properties of Di-Ras1 the that Di-Ras proteins are in GTP-bound physiological Di-Ras has the following substitutions at two positions known to be involved in GTPase activity and conserved in most of other small amino and 65 to and in Ha-Ras, of these amino in its intrinsic GTPase which in of with substitutions have been in other members of the Ras family. For example, has a of highly conserved at position and the GTP-bound form of the protein in HEK293T and cells Y. M. Y. M. T. M. Mol. Cell. Biol. Scholar). some members of family have also been to have substitutions at three positions to be important for GTP hydrolysis Y. J. J. Mol. Cell. Biol. 1996; Scholar, Mol. Cell. Biol. 1998; Scholar, J. Cell Biol. 1998; Scholar). has a at position and to amino and in Ras, has no level of GTPase activity and to be a GTP-bound It is considered that has an on signaling its physiological function to be the amino acid in Di-Ras1 that to Ras for the of its GTPase we performed to amino and 65 with their and in other members of the Ras family. The the intrinsic GTPase activity of not the GTPase activity to the level of that with not These results that these amino acids, other amino acid of Di-Ras1 may also be for its GTPase to characterized proteins, it is expected that the GTP-bound Di-Ras is the active is is the cellular activity of Di-Ras possibility is that Di-Ras function is by proteins or by at specific An possibility is that the Di-Ras activity is at an expression we not Di-Ras1 by using an in some human cell Di-Ras1 be not The to the Di-Ras1 protein may be because of low of the the low level of protein however, it is that the protein level of Di-Ras1 is at the The members of Ras family, at least 13 at present, are characterized by extensive similarities in their effector domains, and many Ras members can interact with the same effector proteins including Raf (4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Google Scholar, J.L. EMBO J. 1998; 17: 6776-6782Google Scholar). As for Di-Ras1 and Di-Ras2, their effector domains are the same as other Ras members, for amino acid 37 corresponding to position in position is important in the Ras family for interaction with the downstream effector Raf, because of Asp-33 to in its interaction with and its activity J. Mol. Cell. Biol. Scholar). we have that Di-Ras of and the also Di-Ras can to the other known Ras effector is known to to the GTP-bound and guanine nucleotide exchange on J. Mol. Cell. Biol. Scholar, Cell. 2001; Scholar). Ha-Ras, we not the interaction of Di-Ras with in a not Although we out the possibility that Di-Ras other effector activated by Ras family members, the novel GTPase might downstream Northern blot of isolated from human that Di-Ras are highly to the brain and expression is to that of a identified Ras-related GTPase, R-Ras3/M-Ras, of which the is also in brain and heart T. M. Oncogene. 1997; Scholar, T. T. Oncogene. 1997; Scholar). has been to have activity in cells M. Oncogene. Scholar, Der C.J. J. Biol. Di-Ras the activity in the cells not and Di-Ras1 may have different biological functions in brain and the expression is have that expression of Di-Ras proteins cellular the of the large by the expression has not been of activated Ras has been known to and a recent report that the stimulation to of the of Cell. 2001; Scholar). however, that a large of the by Di-Ras not a or and Although we out the possibility that the and are because of of the proteins, Di-Ras might be involved in of and their be important for their and functions. The of an important to the biological function of Di-Ras1 and Di-Ras2 have to which has been identified as a suppressor Y. M. Scholar). is expressed in and cells is in and Y. M. Scholar, R. J. Cancer. Scholar). of through growth of and cells Y. M. Scholar). may function as a molecular switch that regulates cell the signaling by the its biochemical to be between Di-Ras and that these proteins can be into a of the Ras family, and they may have roles in cell It would be thus to the possibility that Di-Ras might function as growth in and because these cells are highly and to in In the cellular of small GTPases are through their interaction with or effectors, and have been identified using the and of effector or to Di-Ras would important to of their physiological functions. of the of a novel Ras-related which is to the present was by H. T. Clark G.J. Scholar). for cDNA and for the of