Identification and Characterization of Rain, a Novel Ras-interacting Protein with a Unique Subcellular Localization
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m312867200
ISSN1083-351X
AutoresNatalia Mitin, Melissa B. Ramocki, Alfred J. Zullo, Channing J. Der, Stephen F. Konieczny, Elizabeth J. Taparowsky,
Tópico(s)Phagocytosis and Immune Regulation
ResumoThe Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras. The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras. Ras proteins (H-, N- and K-Ras) are essential components of the extracellular signaling cascades that control cellular proliferation, differentiation, and apoptosis. Mutated alleles of Ras are found in a variety of human tumors (1Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Oncogenic Ras induces the growth and morphological transformation of many cell types in vitro and is directly linked to tumorigenesis in animal models (2Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3792) Google Scholar). Ras is a plasma membrane-localized protein that cycles between GDP-bound (inactive) and GTP-bound (active) states. After activation by external stimuli, Ras-GTP binds with multiple downstream effectors through the core Ras effector-binding domain (residues 32–40), which results in the stimulation of signaling cascades that regulate cytoplasmic and nuclear events. A growing number of Ras effectors have been identified over the years, with the Raf serine/threonine kinases, the phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; RA, Ras-associating; DB, binding domain; GFP, green fluorescent protein; CFP, cyan fluorescent protein; HEK, human embryonic kidney; HA, hemagglutinin; GST, glutathione S-transferase; RBD, Ras-binding domain; RA, Ras-associated; DIL, dilute; YFP, yellow fluorescent protein; GalT, galactosyl transferase. lipid kinases, and the Ral guanine nucleotide exchange factors among the best characterized (3Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (514) Google Scholar, 4Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 5Shields J.M. Pruitt K. McFall A. Shaub A. Der C.J. Trends Cell Biol. 2000; 10: 147-154Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 6Wolthuis R.M. Bos J.L. Curr. Opin. Genet. Dev. 1999; 9: 112-117Crossref PubMed Scopus (128) Google Scholar). Ras activation of effector function is mediated, in part, by the recruitment of these cytoplasmic proteins to the plasma membrane. Recent studies involving live-cell imaging, electron microscopy, and fluorescence resonance energy transfer have shown that in addition to the plasma membrane, H-Ras and N-Ras, but not K-Ras, localize to intracellular membranes of the endoplasmic reticulum and Golgi (7Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Philips and colleagues (8Bivona T.G. Perez de Castro I. Ahearn I.M. Grana T.M. Chiu V.K. Lockyer P.J. Cullen P.J. Pellicer A. Cox A.D. Philips M.R. Nature. 2003; 424: 694-698Crossref PubMed Scopus (361) Google Scholar) have shown that Golgi-localized Ras is activated through a Src/phospholipase Cγ1/Ras guanyl nucleotide-releasing protein pathway that is distinct from the classic protein receptor tyrosine kinase/Grb2/SOS pathway that activates Ras at the plasma membrane. Furthermore, they demonstrated that endomembrane-associated Ras is biologically active because silencing of endogenous Ras guanyl nucleotide-releasing protein-mediated activation of Ras completely blocks T-cell receptor-stimulated Ras activation in Jurkat cells and neuronal differentiation in PC-12 cells. In NIH3T3 transformation assays, a constitutively active, palmitoylation-deficient H-Ras mutant that is localized and activated on endomembranes exhibits transforming activity comparable with that of wild-type activated H-Ras (9Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (522) Google Scholar). Whether the endomembrane and plasma membrane pools of biologically active Ras use the same set of effector molecules remains unknown. Genetically engineered variants of Raf, PI3K, and Ral guanine nucleotide exchange factors that are targeted to the plasma membrane in the absence of Ras activation are constitutively active as signaling molecules (10Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (890) Google Scholar, 11Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (851) Google Scholar, 12Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar, 13Wolthuis R.M. de Ruiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar), establishing these proteins as bona fide effectors of plasma membrane-localized Ras. The identification of the effectors of endomembrane-associated Ras is the next challenge in understanding the role and importance of Ras signaling originating from endomembranes. Herein, we report the identification of Rain, a novel Ras-interacting protein that displays the characteristics of an effector of endomembrane-localized Ras. First, Rain possesses a Ras-associating (RA) domain homologous to the RA domains of other Ras effectors, including the Ral guanine nucleotide exchange factors, AF-6, Rin1, and phospholipase Cϵ (14Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (180) Google Scholar). Second, like Raf and all other known Ras effectors, Rain preferentially binds to the GTP-loaded form of Ras in vitro and in vivo, and the interaction of Rain with Ras is abolished by mutations in the core effector domain of Ras. Third, we show that Rain localizes to a perinuclear, juxta-Golgi region in intact cells and is recruited to the Golgi by active Ras. Finally, we determined that co-expression of Rain together with activated Raf causes synergistic transformation of NIH3T3 cells. These data support a role for Rain as a novel effector for the activated pool of Ras localized to endomembranes in mammalian cells. Yeast Two-hybrid Assays—Individual Gal4 DNA binding domain (DB)-Ras fusion constructs (DB-Ras G12V,C186S; DB-Ras G12V,T35S,C186S; DB-Ras G12V,E37G,C186S; and DB-Ras G12V,Y40C,C186S) were generated by standard procedures. Full-length cDNA sequences encoding effector domain mutants of activated H-Ras G12V were amplified by PCR using primers to introduce 5′-BamHI and 3′-EcoRI sites and to alter the coding sequence to contain a C186S substitution of the cysteine residue of the H-Ras CAAX prenylation signaling motif. The resulting PCR products were cloned into the yeast expression vector pAS2–1 (BD Biosciences Clontech). Two hybrid screens were performed in Saccharomyces cerevisiae strain Y190 using RasG12V,C186S-Gal4 DB as the bait and a human skeletal muscle cDNA library cloned into pACT2 (BD Biosciences Clontech). Transformants were plated on Leu- Trp- His- selection, and large colonies were assayed for β-galactosidase activity as described previously (15Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). The pACT2 plasmid was recovered from each β-galactosidase-positive colony and propagated in Escherichia coli strain DH5. All positive clones were sequenced and characterized further as described in the text. A Gal4 DB-Rain (amino acids 79–447) yeast expression construct in pAS2–1 was generated using available NcoI sites. Interactions between Rain and Ras family members were tested by co-transformation of S. cerevisiae strain L40 (gift from A. Vojtek) with pACT2 Rain 79–447 and individual small GTPases expressed in yeast as LexA DB fusion proteins (gift from A. Vojtek and M. Hansen) (16Vojtek A.B. Hollenberg S.M. Methods Enzymol. 1995; 255: 331-342Crossref PubMed Scopus (243) Google Scholar). Transformants were selected and liquid cultures assayed for β-galactosidase activity as described above. Isolation of the Full-length Rain cDNA—Rain 79–962 (clone C15) was excised from the pACT2 plasmid by EcoRI/XhoI digestion and cloned into pBS-KS. Expressed sequence tag clone AI928221 was obtained from the American Type Culture Collection and used to isolate the cDNA sequence encoding the N terminus of Rain. A 600-bp fragment (encoding amino acids 1–180 of Rain) was isolated by EcoRI/SacI digestion and subcloned into the pBS-KS plasmid (designated Rain 1–180/pBS). pBS Rain 79–962 was digested with SacI, and the isolated 2.2-kb fragment was inserted into the SacI site of pBS Rain 1–180. A plasmid with a confirmed orientation of the SacI fragment then was digested with EcoRI/HincII to excise a 1.1-kb fragment encoding the Rain amino terminus, and this fragment was subcloned into pBS Rain 79–962 digested with EcoRI/HincII to generate a full-length Rain cDNA. This full-length cDNA was subcloned into the pcDNA, pcDNA3 HA, and pBabe-puro mammalian expression vectors. An expression vector encoding Rain tagged at the amino acid terminus with green fluorescent protein Rain (GFP-Rain) was generated by PCR amplification of the enhanced GFP sequence and insertion into pcDNA3 Rain. The same approach was used to generate an expression vector encoding cyan fluorescent protein-tagged Rain (CFP-Rain). The human Rain cDNA sequence reported here has been entered into the GenBank data base under accession number AY378097. Cell Culture and Transient Transfection Assays—NIH3T3 mouse fibroblasts were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. HEK293 cells were maintained in high glucose Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1 mm sodium pyrophosphate, and antibiotics. COS-1 cells were cultured in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. NIH3T3 cells or HEK293 cells (5 × 105 and 3 × 105 cells per 100-mm dish, respectively) were transfected using the standard calcium phosphate DNA precipitation method. Forty-eight hours after transfection, cells were collected in radioimmunoprecipitation assay buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) containing protease inhibitor mixture (Sigma) and whole cell extracts were used for immunoblot analyses. A rabbit polyclonal antibody raised against a carboxyl-terminal human Rain peptide (EQQELPANYRHGPPVATSP; residues 944–962) was used to detect endogenous and exogenously expressed Rain proteins in cell extracts. Hemagglutinin (HA) epitope-tagged Rain was detected with an anti-HA antibody (3F10; Roche Applied Science). NIH3T3 Transformation Assays—We employed a cooperation focus formation assay to assess the contribution of Rain to transformation through Ras signaling pathways. NIH3T3 cells were plated at 1 × 105 cells per 60-mm dish and transfected the next day using LipofectAMINE Plus (Invitrogen) and procedures recommended by the manufacturer. DNA constructs used for transfection were pBabe-raf-Raf22W (50 ng/plate) (17Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar), pcDNA3HA RhoA63L (1 μg/plate) (18Solski P.A. Helms W. Keely P.J. Su L. Der C.J. Cell Growth Differ. 2002; 13: 363-373PubMed Google Scholar), and pcDNA3 Rain (1 μg/plate). Raf22W is an amino-terminally truncated, constitutively activated variant of human Raf-1. RhoA(63L) is a GTPase-deficient, constitutively activated mutant of human RhoA. Cells were fed growth medium 3 h after transfection and every 48 h thereafter. The appearance of transformed foci was monitored for 16 days and quantified visually using phase contrast microscopy. Data are expressed as number of foci per plate, averaged from three plates per group from three independent transfection experiments. For documentation, the cultures were fixed and stained with 0.4% crystal violet. Rain Expression Analyses—To evaluate Rain mRNA expression in the mouse, we used total RNA isolated from adult mouse tissues using TRIzol reagent (Invitrogen) and reverse transcription-PCR. The primers for the mouse Rain cDNA were as follows: 5′-CTACTCTGGGTGTGTTCCAGGC-3′ (forward), 5′-TGCGTCATCTGTCACAGGGC-3′ (reverse) and were designed to amplify a 550-bp fragment at the 3′ end of the Rain open reading frame. Two micrograms of total RNA from each tissue were reverse-transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase for 1 h, and 1 μl of each reaction was amplified by PCR with Taq polymerase for 35 cycles. For human Rain mRNA expression analysis, a multiple-tissue human mRNA blot (BD Biosciences Clontech) was probed with a randomprimed, 32P-labeled 2.1-kb cDNA fragment of Rain (nt 1167–3230). Blots were hybridized at 65 °C in rapid-hybridization buffer (Amersham Biosciences) for 2 h, washed, and exposed to x-ray film. To evaluate Rain protein expression, mouse tissues isolated from an adult FVB mouse were lysed by homogenizing in radioimmunoprecipitation assay buffer containing protease inhibitors. Extracts were cleared by centrifugation and protein concentration determined by Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of extract were separated by SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and immunoblotted using the polyclonal anti-Rain antiserum described above. Interaction between Rain and Small GTPases—Bacterially expressed glutathione S-transferase (GST) fusion proteins containing the isolated Ras-binding domain of c-Raf-1 (amino acids 51–131; Raf-RBD) and a region of Rain containing the RA domain (amino acids 121–245; Rain-RA) were expressed and purified from Rosetta E. coli strain (Novagen, San Diego, CA). Bacterially expressed and purified H-Ras protein (PanVera, Madison, WI) was pre-loaded with 2 mm β,γ-imido-GTP or GDP by incubating for 10 min at 37 °C in 40 μl of loading buffer (20 mm Tris, pH 7.5, 10 mm EDTA, 5 mm MgCl2 and 1 mm dithiothreitol). Reactions were cooled on ice, the MgCl2 concentration adjusted to 15 mm, and the samples diluted to 300 μl with binding buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm MgCl2, and 0.1% Triton-X100) supplemented with 0.2% bovine serum albumin and 25 μm concentration of the corresponding nucleotide before incubation with 5 μg of GST-RA-Rain or GST-Raf-RBD proteins for 2 h at 4 °C. Protein complexes bound to glutathione beads were washed three times with binding buffer, eluted in SDS sample buffer, and resolved by SDS-PAGE. The amount of H-Ras protein bound to RA-Rain or Raf-RBD was detected by immunoblotting using a pan-Ras antibody (Ab-3; Oncogene, San Diego, CA). To determine the ability of Rain to bind to other small GTPases, HEK293 cells were transiently transfected with 2 μg of plasmid DNA expressing Flag epitope-tagged versions of activated H-Ras, Rap1A, R-Ras, or M-Ras/R-Ras3 (a gift of L. Quilliam). Forty-eight hours after transfection, cells were lysed in buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 1 mm EDTA, and 1% Triton X-100) containing protease inhibitors and 250 μg of each cell lysate were incubated for 1.5 h at 4 °C with 5 μg of GST, GST-Raf-RBD, or GST-Rain-RA proteins immobilized on glutathione beads. Protein complexes were washed three times, eluted with SDS sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using an anti-Flag antibody (M2; Sigma). Co-immunoprecipitation Analyses—NIH3T3 cells stably expressing H-Ras61L alone, or together with HA-Rain, were grown to subconfluence and lysed in buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 0.1 mm MgCl2, 10% glycerol, and 0.5% Nonidet P-40) containing protease inhibitors. Five hundred micrograms of total protein extract in a volume of 1 ml were incubated overnight at 4 °C with 50 μl of anti-HA antibody coupled to agarose beads (clone 3F10; Roche Applied Science). Protein complexes were washed three times with lysis buffer, eluted from the beads with SDS sample buffer, and resolved by 12.5% SDS-PAGE. Bound proteins were detected by immunoblot analysis using anti-HA and anti-Ras antibodies. Immunofluorescence Analyses—COS-1 cells were plated at 3 × 105 cells per 60-mm plate and transfected with 2 μg of HA-Ras61L, 3 μg of GFP-Rain, or both constructs using Superfect transfection reagent (QIAGEN). Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde and stained using an anti-HA antibody (HA.11; Covance Inc., Princeton, NJ) as described previously (19Mitin N. Ramocki M.B. Konieczny S.F. Taparowsky E.J. Methods Enzymol. 2001; 333: 232-247Crossref PubMed Scopus (4) Google Scholar). Staining was visualized using an Olympus fluorescence microscope with a 60× objective. To confirm Rain localization at the Golgi, 1 × 105 COS-1 cells were plated onto 35-mm MatTek dishes containing number 1.5 glass slides on the bottom (MatTek Corp., Ashland, MA) and transfected as described above with 1 μg of CFP-Rain and 0.3 μg of YFP-GalT (a gift from M. Philips) in the presence or absence of 0.5 μg of H-Ras61L-Flag. Twenty-four hours after transfection, cells were fixed in 4% paraformaldehyde or viewed live using a Zeiss 510 LSM confocal microscope. Identification of a Novel Ras-interacting Protein—To identify additional Ras effectors, a yeast two-hybrid screen was performed using a Gal4-DB-H-Ras G12V fusion protein as the bait and an adult human skeletal muscle cDNA library as the source of interacting proteins. From 1.8 × 107 independent transformants, 46 clones were selected that displayed interaction dependent growth on His- agar and high levels of β-galactosidase activity. The specificity of the interaction with Ras G12V was confirmed by retesting each clone with a variety of control baits (data not shown). Sequence analysis of the clones revealed that 41 represented partial cDNA sequences for the known Ras effectors, Rin1, RalGDS, and RalGDS-like 2 protein. The remaining five clones represented cDNAs encoding the same protein. All of these cDNAs were 2.9 kb long and contained an open reading frame of 883 amino acids, a 284-bp 3′ untranslated region, and a poly(A) tail. A search of the GenBank nucleotide data base with the sequence of a representative clone (C15) revealed no significant homology with any known genes but identified several expressed sequence tag clones identical to regions of the C15 cDNA. One expressed sequence tag (GenBank accession number AI928221) contained information that was used to extend the C15 cDNA by 0.3 kb in the 5′ direction and to incorporate an in-frame initiator ATG codon (Fig. 1A). The completed cDNA (3.2 kb) is identical to a recently released IMAGE clone (GenBank accession number BC028614) and contains an open reading frame for a 962-amino acid, highly basic (pI = 8.7) polypeptide with a predicted molecular mass of 104 kDa. Because the protein encoded by this cDNA binds to Ras, we named this protein Rain, for Ras-interacting protein. Rain contains a single RA (RalGDS/AF-6) domain (14Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (180) Google Scholar) in the N terminus (Fig. 1A). This domain (amino acids 144–247) has the highest homology to the RA domains of human AF-6 (Fig. 2), a protein first identified as a 3′ portion of a translocation product in some human leukemias (20Prasad R. Gu Y. Alder H. Nakamura T. Canaani O. Saito H. Huebner K. Gale R.P. Nowell P.C. Kuriyama K. et al.Cancer Res. 1993; 53: 5624-5628PubMed Google Scholar) and subsequently as a putative Ras/Rap effector (21Van Aelst L. White M.A. Wigler M.H. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 181-186Crossref PubMed Scopus (53) Google Scholar). An amino acid sequence alignment of the RA domain of Rain with the RA domains of other known Ras-interacting proteins is shown in Fig. 1B. Rain possesses the conserved basic residue (Arg182) found to be critical for the interaction of other effectors with Ras [Arg-89 in c-Raf-1 and Lys-685 in RGL (22Fabian J.R. Vojtek A.B. Cooper J.A. Morrison D.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5982-5986Crossref PubMed Scopus (158) Google Scholar, 23Shirouzu M. Hashimoto K. Kikuchi A. Yokoyama S. Biochemistry. 1999; 38: 5103-5110Crossref PubMed Scopus (12) Google Scholar)]. In addition to the RA domain, Rain contains a proline-rich region (amino acids 59–136) that is similar to the Src homology 3 domain binding motifs found in many intracellular proteins. Rain also contains a putative dilute (DIL) domain (amino acids 769–877) found in the globular tail of the myosin V proteins (24Ponting C.P. Trends Biochem. Sci. 1995; 20: 265-266Abstract Full Text PDF PubMed Scopus (68) Google Scholar). In myosins, the DIL domain may be involved in binding to cargo during vesicle trafficking (25Rogers S.L. Karcher R.L. Roland J.T. Minin A.A. Steffen W. Gelfand V.I. J. Cell Biol. 1999; 146: 1265-1276Crossref PubMed Scopus (110) Google Scholar, 26Wu X. Bowers B. Rao K. Wei Q. Hammer J.A.R. J. Cell Biol. 1998; 143: 1899-1918Crossref PubMed Scopus (342) Google Scholar). It is interesting that, outside of the myosin V family, AF-6 is the only protein found to contain a DIL domain. However, the role of this domain in AF-6 function is unknown. When the amino acid sequence of Rain was used to search the NCBI protein data base, we identified a protein sequence that is likely to represent a rat homolog of Rain (accession number XP_214916). In addition, this search identified a second human and mouse protein pair (accession number NP_060529 and NP_84887, respectively) that has the same domain architecture as Rain. These proteins, along with the AF-6 and its orthologs, are likely to comprise a new family of proteins with a unique combination of protein domains (Fig. 2). No Rain orthologs have been identified in invertebrates. Our search of the human high throughput genomic sequence data base identified two genomic contigs containing the Rain gene (GenBank™ accession numbers AC009002 and AC008888). The contigs and the hypothetical protein they encode (FLJ20401) map to chromosome 19q13.33. Alignment of the Rain cDNA with its genomic sequence revealed that the Rain gene consists of 12 exons and 11 introns, 10 of which interrupt the protein-coding region (Fig. 1C). Expression analysis of the human RAIN gene revealed a single transcript (∼3.5 kb) that closely matched the size of the cloned Rain cDNA in all human tissues examined, with highest levels found in heart (Fig. 3A). Reverse transcription-PCR analysis of RNA isolated from adult mouse tissues confirmed that the Rain gene is transcriptionally active in the majority of samples examined, with the highest level of Rain mRNA detected in mouse lung (Fig. 3B). To evaluate the expression of the Rain protein, we used a carboxyl-terminal peptide from human Rain to generate a rabbit polyclonal antiserum. Immunoblot analysis of protein extracts prepared from mouse tissues with the anti-Rain antiserum revealed two peptides of 115 and 85 kDa in lung and a single 85-kDa peptide in spleen (Fig. 3C). To test the specificity of the Rain antiserum, immunoblots were performed with protein extracts isolated from mouse lung or from NIH3T3 cells expressing HA-tagged Rain. The anti-HA antibody detected the 115-kDa HA-Rain protein expressed in the transfected cells (Fig. 3D). When the same extracts were immunoblotted using anti-Rain antibodies, 115- and 85-kDa peptides were detected in both samples. Pre-incubation of the anti-Rain antibodies with the Rain peptide used to generate the antiserum completely abolished detection of the 115-kDa protein and had no effect on the detection of the 85-kDa peptide. No proteins of 115 kDa were observed when pre-immune rabbit serum was used to probe the same samples (data not shown). Thus, together with the predicted size of Rain, these analyses indicate that the 115-kDa band corresponds to endogenous mouse Rain protein. Rain Binds to Ras in a GTP-dependent Manner—Ras effectors preferentially bind to the activated (GTP-bound) form of Ras through a region referred to as the core effector domain (Ras residues 32–40). Yeast two-hybrid assays were used to evaluate Rain interaction with different Ras effector domain mutants. These results show that an intact Ras effector domain is necessary for interaction with Rain. Although Rain showed a strong interaction with E37G H-Ras effector domain variant, no interaction was detected using the T35S or Y40C variants (Fig. 4A). We next determined whether the Rain-Ras interaction is influenced by the activation state of Ras. Recombinant Ras protein was loaded with GDP or β,γ-imido-GTP (a non-hydrolyzable GTP analog) and incubated with a GST fusion protein containing the isolated RA domain of Rain (amino acids 121–245). The RBD of Raf-1 (Raf-RBD) was used as a positive control. The amount of Ras protein bound was determined by immunoblotting using an anti-Ras antibody. As shown in Fig. 4B, Rain exhibited a higher affinity for Ras-GTP than Ras-GDP. As expected, a similar binding preference was observed for the Raf-RBD. When we evaluated the ability of Ras to bind to Rain peptides lacking the RA domain, no interactions were detected (data not shown), indicating that the RA domain is necessary and sufficient for interaction with Ras. Taken together with the yeast two-hybrid studies, these results show that Rain meets the criteria of an effector for Ras. We next tested whether the Rain-Ras interaction occurs in mammalian cells. For these studies, NIH3T3 cells were stably transfected with an expression vector for activated H-Ras (Ras61L) and either a control plasmid or a plasmid expressing full-length, HA-tagged Rain. HA-Rain was immunoprecipitated from whole cell lysates using an anti-HA antibody, and the protein complexes were resolved by SDS-PAGE and immunoblotted using an anti-Ras antibody. Results revealed that the Ras protein is detected only in precipitates from HA-Rain-expressing cells (Fig. 4C). These results demonstrate that Rain-Ras interaction occurs in mammalian cells and supports a role for Rain as a physiologically relevant effector of Ras. Rain Binds to Other Ras Family Members—Because many of the Ras effectors characterized to date (e.g. Raf, RalGDS, AF-6, Nore1) interact with other Ras-related proteins in addition to Ras, we used yeast two-hybrid assays to test the ability of the Rain RA domain to bind to other members of the Ras superfamily of GTPases. In agreement with our initial studies with the Gal4 DB-Ras G12V bait, Rain interacted with the LexA DB-H-Ras G12V fusion protein (Fig. 4D). Rain also interacted strongly with Rap2 and with activated R-Ras, Rap1A and TC21/R-Ras2 proteins. No interaction was det
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