Identification and Characterization of a Novel Protein Interacting with Ral-binding Protein 1, a Putative Effector Protein of Ral*

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.

Ral is a member of small G protein 1 superfamily and consists of RalA and RalB (1,2). As well as other small G proteins, Ral has the GDP-bound inactive and the GTP-bound active forms. The GDP-bound form of Ral is converted to the GTP-bound form by RalGDS, and inversely the GTP-bound form is changed to the GDP-bound form by RalGAP (3,4). We and other groups have found that RalGDS is a putative effector protein of Ras (5)(6)(7). Since RalGDS stimulates the GDP/GTP exchange of Ral (4), it is possible that there is a new signaling pathway from Ras to Ral through RalGDS. Indeed, it has been shown that RalGDS stimulates the GDP/GTP exchange of Ral in a Ras-dependent manner in COS cells and that a dominant negative form of Ral blocks a Ras-dependent transformation in NIH3T3 cells (8). It has been also demonstrated that Ral is required for Src-and Ras-dependent activation of phospholipase D and that it regulates the initiation of border cell migration induced by Ras in Drosophila oogenesis (9,10). Furthermore, it has been shown that RalGDS and Raf synergistically stimulate cellular proliferation and gene expression (11,12). These results indicate that RalGDS and Ral act downstream of Ras and mediate Ras functions. However, the mechanism by which Ral regulates cellular functions is not known.
One possible clue to clarify the mode of action of Ral is RalBP1 (13)(14)(15). RalBP1 has a Ral-binding domain in its Cterminal region and binds to the GTP-bound form of Ral but not to the GDP-bound form. A mutation in the effector loop of Ral impairs its interaction with RalBP1 and RalBP1 inhibits the RalGAP activity for Ral (13,16). These results suggest that RalBP1 is an effector protein of Ral. RalBP1 also has a RhoGAP homology domain in its central region and exhibits the GAP activity for Rac1 and CDC42 but not for RhoA (13)(14)(15). Recently we have found that Ral localized to the membrane through its post-translational modification induces translocation of RalBP1 from the cytosol to the membrane and that the posttranslational modifications of Rac1 and CDC42 enhance the GAP activity of RalBP1 (17). Therefore, RalBP1 may link Ral to Rac or CDC42 on the membrane. However, there is no evidence obtained so far that RalBP1 regulates the activities of Rac and CDC42 in intact cells. It is not known whether RalBP1 has additional activities.
To gain more insight into the action of RalBP1, we sought to isolate proteins which interact with RalBP1 by yeast two-hybrid screening. We describe here the isolation of a novel RalBP1-interacting protein, which is designated POB1 (partner of RalBP1). POB1 shares a homology with Eps15, which has been identified as an EGF receptor substrate (18), and has two proline-rich motifs. POB1 and Ral simultaneously interact with RalBP1 on the different sites and these proteins make a ternary complex. Furthermore, POB1 binds to Grb2, is tyrosine-phosphorylated in COS cells in response to EGF, and makes a complex with EGF receptor. These results suggest that RalBP1 and POB1 may provide a link between tyrosine kinase, SH3-containing protein, and Ral.

EXPERIMENTAL PROCEDURES
Materials and Chemicals-Yeast strain L40, plasmid vectors for two-hybrid screening, pGAD10-derived human brain cDNA library, and pEF-BOS were kindly supplied from Drs. Y. Takai, K. Tanaka, and S. Nagata (Osaka University, Suita, Japan) (19,20). gt10 human brain cDNA library was from Dr. K. Kaibuchi (Nara Institute Science and Technology, Ikoma, Japan) (21). pGEX/Grb2 was a generous gift from * This work was supported by grants-in-aid for scientific research (B) and for scientific research on priority areas from the Ministry of Education, Science, and Culture, Japan (1996, 1997) and by grants from Yamanouchi Foundation for Research on Metabolic Disorders (1996), Fukuyama Transporting Shibuya Longevity and Health Foundation (1996), and Kato Memorial Bioscience Foundation (1997). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF010233.
Drs. H. Miki and T. Takenawa (Institute of Medical Science, University of Tokyo, Tokyo, Japan) (22,23). pBSSK/Nck and pGFP/Crk were generous gifts from Drs. H. Hanafusa (Rockefeller University, New York) and M. Matsuda (National Institute of Health, Tokyo, Japan) (24), respectively. The anti-GST and Ral rabbit polyclonal antibodies were made by a routine method and supplied from Dr. M. Nakata (Sumitomo Electric Industries, Yokohama, Japan). The anti-HA antibody 12CA5 was kindly provided by Dr. Q. Hu (Chiron Corp., Emeryville, CA). The anti-Myc antibody was prepared from 9E10 cells. RalBP1 cDNA was synthesized by reverse-transcriptase PCR as described (16). [␣-32 P]dCTP, [ 35 S]GTP␥S, and [␥-32 P]GTP were purchased from Amersham Inc. (Buckinghamshire, United Kingdom). The antiphosphotyrosine and EGF receptor antibodies were from ICN Biomedicals, Inc. (Costa Mesa, CA) and Transduction Laboratories (Lexington, KY), respectively. EGF was purchased from Life Technologies, Inc. Other materials were from commercial sources.
Yeast Two-hybrid Screening-Yeast strain L40 (MATa trp1 leu2 his3 ade2 LYS2::lexA-HIS3 URA3::lexA-lacZ) was used as a host for the two-hybrid screening (27). Yeast cells were grown on rich medium (YAPD) containing 2% glucose, 2% Bacto-peptone, 1% Bacto-yeast extracts, and 0.002% adenine sulfate. Yeast transformations were performed by the lithium acetate method. Transformants were selected on SD medium containing 2% glucose, 0.67% yeast nitrogen base without amino acids, and appropriate amino acids, which were supplemented to SD medium when required. A strain L40 carrying pBTM116HA/RalBP1 was transformed with a human brain cDNA library constructed in pGAD10. pBTM116HA/RalBP1 directs the expression of a fusion between the DNA-binding domain of LexA and the entire RalBP1 from an ADH promoter. Approximately 6 ϫ 10 6 transformants were screened for the growth on SD plate medium lacking tryptophan, leucine, and histidine as evidenced by transactivation of a LexA-HIS3 reporter gene and histidine prototrophy. His ϩ colonies were scored for ␤-galactosidase activity. Plasmids harboring cDNAs were recovered from positive colo-nies and introduced by electroporation into E. coli HB101 on the M9 plate lacking leucine. HB101 is leuB Ϫ , and this defect can be complemented by the LEU2 gene in the library plasmid. Then library plasmids were recovered from HB101 and transformed again into L40 containing pBTM116HA/RalBP1. The nucleotide sequences of plasmid DNAs, which conferred the LacZ ϩ phenotype on L40 containing pBTM116HA/ RalBP1, were determined.
Molecular Cloning of POB1-The clone identified by the yeast twohybrid method contained a region that interacted with RalBP1 and the 3Ј-end but did not contain the 5Ј-end of an open reading frame. To obtain full-length cDNA, the clone was labeled with random primers and [␣- 32 P]dCTP and used to screen gt10 human brain cDNA library. However, the initial screening did not identify the 5Ј-end of the gene. To obtain more 5Ј sequence information, the 5Ј-most 650 nucleotides of the largest clone was labeled with [␣-32 P]dCTP to screen the same library, and a number of positive clones were isolated. All clones, collectively spanning 3.2 kb, were sequenced using double-strand templates and Thermo Sequenase premixed cycle sequencing kit (Amersham, Buckinghamshire, UK) and Hitachi DNA sequencer SQ-5500 (Hitachi Ltd., Tokyo, Japan). To construct full-length POB1 cDNA clone, the cDNA fragment containing POB1 codons 10 -521 (amino acid numbers) from the initial screening was subcloned into pUC19. The cDNA fragment containing POB1 codons 1-253 (amino acid numbers) from the second screening was jointed to this sequence to obtain pUC19/POB1.
Northern Blot Analysis-Total RNA was extracted from various rat tissues as described (28). Northern blot analysis was performed as described (29). Twenty g of total RNA was then resolved by agarose gel electrophoresis and transferred to nitrocellulose filters. A 1.6-kb cDNA probe corresponding to full-length POB1 was labeled with random primers and [␣-32 P]dCTP and hybridized to the membrane. The membrane was washed and exposed to Kodak X-Omat film.
Interaction of POB1 and Ral with RalBP1 in Vitro-Various MBP fused to RalBP1 deletion mutants (20 pmol each) were incubated with GST-POB1-(375-521) (44 pmol) or the GTP␥S-bound form of GST-Ral (44 pmol) in 40 l of reaction mixture (20 mM Tris/HCl (pH 7.5), 1 mM dithiothreitol, and 0.05% CHAPS) for 1 h at 4°C. The GTP␥S-bound form of Ral was made as described (16). The amylose resin was added to this mixture and further incubation was performed. After 30 min, the resin was precipitated by centrifugation. The precipitates were probed with the anti-GST antibody.
Interaction of POB1 with Grb2, Nck, and Crk in Vitro-The lysates (465 g of protein) of COS cells expressing HA-POB1 were incubated with 1 M GST-Grb2, GST-Nck, GST-Crk, or GST in 170 l of the lysis buffer for 1 h at 4°C. GST fused to proteins was precipitated with glutathione-Sepharose 4B. The precipitates were probed with the anti-HA and GST antibodies.
Complex Formation of POB1 with EGF Receptor-COS cells express-ing HA-POB1 with or without Myc-RalBP1 were serum-starved for 24 h before stimulation with 100 ng/ml EGF for 15 min. The lysates (620 g of protein) were prepared as described above except that 5 mM sodium orthovanadate and 50 mM ␤-glycerophosphate were added to the lysis buffer and immunoprecipitated with the anti-HA antibody. The precipitates were probed with the anti-HA, Myc, phosphotyrosine, or EGF receptor antibody.
Other Methods-The assay for the interaction of the [ 35 S]GTP␥Sbound form of Ral with immobilized MBP-RalBP1 in the presence of GST-POB1-(375-521) was carried out as described (17). The GAP activity of RalBP1 for the post-translationally modified Rac1 was measured in the presence of GST-POB1-(375-521) as described (17). Protein concentrations were determined using bovine serum albumin as a standard (32).

RESULTS
Isolation of RalBP1-interacting Protein-To identify proteins that physically interact with RalBP1, we conducted a human brain cDNA library screening with the yeast two-hybrid method. From 6 ϫ 10 6 initial transformants, we identified five positive clones (His ϩ and LacZ ϩ ) and the library plasmids were recovered from the clones. Among these five plasmids, three clones were found to confer both the His ϩ and LacZ ϩ phenotypes on L40 containing pBTM116HA/RalBP1. The cDNA inserts from these three clones were 1 kb, and all encoded a single sequence containing an open reading frame of 147 amino acids and the consensus sequence for a stop codon.
To identify full-length cDNA of this RalBP1-interacting protein, a human brain cDNA library was screened using the cDNA isolated by the yeast two-hybrid method and a number of overlapping positive clones were isolated. The overlapping clones spanned a distance of 3.2 kb and contained an uninterrupted open reading frame of 1563 base pairs, encoding a predicted protein of 521 amino acids with a calculated M r of 57,901 (Fig. 1A). We designated this protein POB1 for partner of RalBP1. The first ATG was preceded by stop codons in all three reading frames and the 5Ј-noncoding region had a high percentage of GC base pairs (72%). The second amino acid was also Met. The neighboring sequence of the second ATG was consistent with the translation initiation start proposed by Kozak (33) better than that of the first ATG, but we could not determine which is the first Met. Data bases were searched for related proteins. Besides short human EST sequences (Gen-Bank™ accession numbers; R90985, H44097, T72336, T72376, R88729, H44028, W38942, G27820, N89827, and AA191588), no protein closely related to POB1 was identified. However, the central region of POB1, residues 126 -227, shares 37% amino acid identity with residues 111-211 of Eps15, which has been identified as an EGF receptor substrate (18) (Fig. 1, B and C). The C-terminal region has two proline-rich motifs, PPTPPPRP (amino acids 338 -345) and PPPPALPPRP (amino acids 374 -383), and a putative coiled-coil structure (Fig. 1B).
By Northern blot analysis, a single band was strongly detected in RNA of rat cerebrum, cerebellum, lung, and testis, and weakly in kidney, but not in heart, thymus, liver, spleen, or adrenal gland (Fig. 2). This tissue distribution of POB1 was different from that of RalBP1, which is ubiquitously expressed in rat various tissues (13,15).
These results indicate that POB1-(375-521) is necessary and sufficient for the binding of POB1 to RalBP1.
Ternary Complex Formation of Ral, RalBP1, and POB1-To examine whether the interaction of RalBP1 with POB1 affects the Ral binding activity of RalBP1, POB1-(375-521) and the [ 35 S]GTP␥S-bound form of Ral were incubated with immobilized RalBP1. One M [ 35 S]GTP␥S-bound form of Ral was used in this experiment. In the absence of POB1-(375-521), Ral bound to RalBP1 at a molar ratio 0.8:1 (17). POB1-(375-521) did not affect the interaction of Ral with RalBP1, even though 9-fold excess of POB1-(375-521) was added (Fig. 5A). Under these conditions, POB1-(375-521) bound to RalBP1 in a dosedependent manner (Fig. 5A, inset). To clarify the interaction of these three proteins in intact cells, Myc-RalBP1 and HA-POB1 were expressed with HA-Ral G23V , an active form, or HA-Ral S28N , a dominant negative form, in COS cells (Fig. 5B, lanes   FIG. 3. Interaction of POB1 (lanes 1-6) were incubated with GST-POB1-(375-521) (44 pmol), then the amylose resin was added. After the resin was precipitated by centrifugation, the precipitates were probed with the anti-GST antibody. C, interaction of Ral with RalBP1-(391-499). Various MBP-RalBP1 deletion mutants (20 pmol each) (lanes 1-6) described in A (lanes 1-6) were incubated with the GTP␥S-bound form of GST-RalB (44 pmol), then the amylose resin was added. After the resin was precipitated by centrifugation, the precipitates were probed with the anti-GST antibody. The results shown are representative of three independent experiments. 1 and 2). When Myc-RalBP1 was immunoprecipitated with the anti-Myc antibody, both HA-POB1 and HA-Ral G23V were coprecipitated with Myc-RalBP1 (Fig. 5B, lane 3). HA-POB1 but not HA-Ral S28N was coprecipitated with Myc-RalBP1 (Fig. 5B, lane  4). The binding of Ral to RalBP1 was not found to affect that of POB1 to RalBP1. RalBP1 and Ral G23V tagged with HA and Myc epitopes, respectively, were constructed, and further experiments to show the ternary complex formation of Ral, RalBP1, and POB1 were carried out. When Myc-Ral G23V was immunoprecipitated with the anti-Myc antibody from the lysates expressing HA-RalBP1, HA-POB1, and Myc-Ral G23V , both HA-RalBP1 and HA-POB1 were detected in the Ral immune complex (Fig. 5B, lanes 5 and 8). However, when the lysates expressing HA-RalBP1 and HA-POB1 without Myc-Ral G23V or the lysates expressing HA-POB1 and Myc-Ral G23V without HA-RalBP1 were immunoprecipitated with the anti-Myc antibody, HA-POB1 was not precipitated in these immune complexes (Fig. 5B, lanes 6, 7, 9, and 10). These results indicate that the binding of POB1 to RalBP1 does not affect the Ral-binding activity of RalBP1, that activated Ral and POB1 can simulta-neously interact with RalBP1, and that these proteins make a ternary complex in intact cells.
Effect of the Interaction of POB1 with RalBP1 on the GAP Activity of RalBP1-It has been shown that RalBP1 possesses the GAP activity for Rac and CDC42 (13)(14)(15) and that the binding of Ral to RalBP1 does not affect the GAP activity (15,17). The effect of the interaction of POB1 with RalBP1 on the GAP activity was examined. RalBP1 stimulated the GTPase activity of Rac in a dose-dependent manner (Fig. 6). The GAP activity of RalBP1 for Rac was not affected by the interaction of POB1-(375-521) (Fig. 6).
Interaction of POB1 with Grb2-POB1 has two proline-rich motifs, PPTPPPRP 345 and PPPPALPPRP 383 (Fig. 1A). These amino acid sequences belong to class II ligands of proline-rich motifs, which are known to associate with SH3 domains of adaptor proteins such as Grb2, Nck, and Crk (34,35). To examine the possibility of the interaction of POB1 with these adaptor proteins, Grb2, Nck, and Crk were purified as GST fused to proteins. The lysates of COS cells expressing HA-POB1 were incubated with GST-Grb2, GST-Nck, GST-Crk, or GST. POB1 bound to Grb2 specifically among these proteins (Fig. 7).
Complex Formation of POB1 with EGF Receptor-Besides proline-rich motifs, POB1 has a homologous region with Eps15, which has been identified as an EGF receptor substrate (Fig.  1). Therefore, it was examined whether POB1 is involved in tyrosine kinase signaling through EGF receptor. When COS cells expressing HA-POB1 were stimulated with EGF, HA-POB1 was tyrosine-phosphorylated (Fig. 8A). This tyrosine phosphorylation did not affect the interaction of HA-POB1 with Myc-RalBP1 in intact cells (Fig. 8B). Furthermore, HA-POB1 was immunoprecipitated with a tyrosine-phosphorylated 170-kDa protein in response to EGF, and this protein was recognized by the anti-EGF receptor antibody (Fig. 8C). Taken together, these results indicate that POB1 is tyrosinephosphorylated in response to EGF and that it may make a complex with EGF receptor through an adaptor protein such as Grb2. DISCUSSION We have isolated here a protein interacting with RalBP1 and determined its primary structure. This protein is a novel protein and named POB1. We have found that the C-terminal region (amino acids 375-521) of POB1 is important for its binding to RalBP1 using intact cell and cell-free systems. Furthermore, we have shown that the POB1-binding domain of RalBP1 is distinct from the Ral-binding domain, that POB1 does not compete with the GTP␥S-bound form of Ral for binding to RalBP1, and that RalBP1 and POB1 make a complex with an activated form of Ral but not with a dominant negative form of Ral. These results suggest that POB1 and RalBP1 make a binary complex in resting conditions and that POB1- RalBP1 complex binds to Ral when Ral is activated. Sequence analysis reveals that the RalBP1-binding domain of POB1 and the POB1-binding domain of RalBP1 have high probabilities of forming coiled coil (14,36). Therefore, the interaction between POB1 and RalBP1 may be mediated by these coiled-coil structures. Although we do not know the physiological functions of POB1 at present, POB1 could be involved in the actions of Ral and RalBP1. RalBP1 is known to possess the GAP activity for Rac and CDC42 (13)(14)(15). The interaction of POB1 with RalBP1 does not affect its GAP activity for Rac1. It has been also shown that the binding of Ral to RalBP1 does not affect its GAP activity (15,17). Therefore, the mechanism by which RalBP1 GAP activity is regulated is still unclear. Northern blot analysis has revealed that the RalBP1 mRNA is present in various rat tissues (13,15), including not only tissues where the POB1 mRNA is detectable but also tissues where it is not detectable. For instance, the RalBP1 mRNA, but not the POB1 mRNA, is present in heart, liver, and spleen. These results raise the possibility that RalBP1 interacts with another protein in the tissues where POB1 is absent.
POB1 has two proline-rich motifs, PPTPPPRP (amino acids 338 -345) and PPPPALPPRP (amino acids 374 -383). Prolinerich motifs have been described as general cognate ligands for numerous SH3-containing proteins (37). The ligands can bind to SH3 domains in either an amino-carboxyl or carboxyl-amino orientation and are therefore classified as class I and class II (34,35). Class I ligands contain (R/K/X)PXPPXP and bind to SH3 domains of Src, Abl, Fyn, and Lyn. Proline-rich motifs of POB1 belong to class II ligands, whose general form is PXX-PXR. The class II ligands are the sites for SH3 domains of adaptor proteins such as Grb2, Nck, and Crk. We have found that among these adaptor proteins POB1 interacts with Grb2 specifically. Grb2 plays a central role in signaling by receptor tyrosine kinase, where its SH2 domain binds to the receptor and its two SH3 domains link to other signaling proteins such as SOS, Vav, c-Abl, and dynamin (38). These results raise the possibility that POB1 may make a complex with the receptor tyrosine kinase. Besides proline-rich motifs, POB1 has a homologous region with Eps15, which has been identified as an EGF receptor substrate (18). As to the relationship between POB1 and EGF receptor, we have found that POB1 is tyrosinephosphorylated in response to EGF and that it makes a complex with EGF receptor. Taken together, these results suggest that POB1 may be recruited to EGF receptor through an adaptor protein (Grb2 is a candidate). We have not yet determined here the tyrosine phosphorylation site of POB1. Although POB1 has two consensus tyrosine sites, Tyr 146 and Tyr 229 , for the phosphorylation by EGF receptor (39), it remains to be clarified whether EGF receptor phosphorylates POB1 directly or other tyrosine kinase does it. This tyrosine phosphorylation of POB1 does not affect its interaction with RalBP1. Therefore, the physiological significance of this phosphorylation also remains to be elucidated.
We have previously demonstrated that EGF induces the interaction of Ras with RalGDS and that activated Ras translocates RalGDS from the cytosol to the membrane (16,30). We have also shown that EGF activates Ral through RalGDS and that activated Ral induces the translocation of RalBP1 (17,40). It is not known whether POB1 acts upstream or downstream of RalBP1. If POB1 works as an upstream molecule of RalBP1, it may transmit the signal from EGF receptor to RalBP1 and collaborate with Ral to regulate the RalBP1 functions. If POB1 works as a downstream molecule of RalBP1, the Ral-RalBP1 complex may regulate the POB1 functions in synergy with the EGF receptor signaling. In either case, the ternary complex of Ral, RalBP1, and POB1 could mediate the tyrosine kinase and Ras signals to downstream events.
Eps15 consists of three domains, I, II, and III. Domain I (amino acids 9 -314) is composed of three nonidentical repeats of about 70 amino acids each. The individual repeats are named EH domain (for Eps15 homology) 1, 2, and 3 (18,41). POB1-(143-208) shares 30, 39, and 33% identity with EH domains 1, 2, and 3, respectively. EH domain of Eps15 has been suggested to be tyrosine-phosphorylated by EGF receptor, and overexpression of Eps15 can transform NIH3T3 cells (18). EH domain has been shown to bind to cytosolic proteins in normal and malignant cells, although they have not yet been identified (41). EH domain is conserved in Eps15r, yeast PAN1, yeast END3, and Caenorhabditis elegans YNJ6 (41). PAN1 in yeast is required for normal organization of the actin cytoskeleton and associates with the actin patches on the cell cortex (42). END3 is required for the internalization step of endocytosis and for actin cytoskeleton organization (43). It has been shown that Ral is localized to synaptic vesicles in neuron and ␣-granules in platelets (44,45) and that RalBP1 exhibits GAP activity for Rac and CDC42, which control cytoskeleton (46). Therefore, it is intriguing to speculate that Ral and RalBP1 regulate endocytosis and cytoskeleton organization through POB1. The study to isolate the protein interacting with EH domain of POB1 is under way. Further studies are necessary to understand the whole picture of the functions of a Ras-signaling pathway through RalGDS, Ral, and RalBP1.