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Interaction of POB1, a Downstream Molecule of Small G Protein Ral, with PAG2, a Paxillin-binding Protein, Is Involved in Cell Migration*

Open AccessPublished:July 30, 2002DOI:https://doi.org/10.1074/jbc.M203453200
      POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl-terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl-terminal region including the SH3 domain of PAG2. Substitutions of Pro423 and Pro426 with Ala (POB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.
      EH
      Eps15 homology
      G protein
      GTP-binding protein
      EGF
      epidermal growth factor
      SH3
      Src homology 3
      Arf
      ADP-ribosylation factor
      GAP
      GTPase-activating protein
      GST
      glutathione S-transferase
      CHO-IR
      Chinese hamster ovary-insulin receptor
      MBP
      maltose-binding protein
      HA
      hemagglutinin
      SPR
      surface plasmon resonance
      BSA
      bovine serum albumin
      PBS
      phosphate buffered saline
      GFP
      green fluorescent protein
      We have identified an EH1 domain-containing protein that binds to RalBP1, which is an effector protein of small G protein Ral (
      • Feig L.A.
      • Urano T.
      • Cantor S.
      ), and named this protein POB1 (
      • Ikeda M.
      • Ishida O.
      • Hinoi T.
      • Kishida S.
      • Kikuchi A.
      ). POB1 has a single EH domain in its amino-terminal region and proline-rich motifs and a coiled-coil structure in its carboxyl-terminal region. The EH domain has been initially identified as a repeated sequence present in three copies in the amino-terminal region of Eps15 and of the related molecule Eps15R, two substrates of the EGF receptor kinase (
      • Fazioli F.
      • Minichiello L.
      • Matoskova B.
      • Wong W.T.
      • Di Fiore P.P.
      ,
      • Coda L.
      • Salcini A.E.
      • Confalonieri S.
      • Pelicci G.
      • Sorkina T.
      • Sorkin A.
      • Pelicci P.G.
      • Di Fiore P.P.
      ). Reps1 (for RalBP1-associated Eps-homology domain protein), intersectin, Pan1, and End3 contain the EH domain in addition to Eps15, Eps15R, and POB1 (
      • Santolini E.
      • Salcini A.E.
      • Kay B.K.
      • Yamabhai M.
      • Di Fiore P.P.
      ). Eps15 binds to α-adaptin, a subunit of the clathrin adaptor complex, AP-2 (
      • Iannolo G.
      • Salcini A.E.
      • Gaidarov I.
      • Goodman O.B.J.
      • Baulida J.
      • Carpenter G.
      • Pelicci P.G., Di
      • Fiore P.P.
      • Keen J.H.
      ). The AP-2-binding site of Eps15 acts dominant negatively in blocking the endocytosis of EGF, transferrin, or Sindbis virus, indicating that Eps15 is actively required for the endocytic process (
      • Carbone R.
      • Fre S.
      • Iannolo G.
      • Belleudi F.
      • Mancini P.
      • Pelicci P.G.
      • Torrisi M.R.
      • Di Fiore P.P.
      ,
      • Benmerah A.
      • Lamaze C.
      • Begue B.
      • Schmid S.L.
      • Dautry-Varsat A.
      • Cerf-Bensussan N.
      ). Eps15R has a 47% amino acid identity with and exhibits similar characteristics to Eps15 (
      • Coda L.
      • Salcini A.E.
      • Confalonieri S.
      • Pelicci G.
      • Sorkina T.
      • Sorkin A.
      • Pelicci P.G.
      • Di Fiore P.P.
      ,
      • Wong W.T.
      • Schumacher C.
      • Salcini A.E.
      • Romano A.
      • Castagnino P.
      • Pelicci P.G.
      • Di Fiore P.P.
      ). The POB1-related protein Reps1 has been identified as a RalBP1-binding protein (
      • Yamaguchi A.
      • Urano T.
      • Goi T.
      • Feig L.A.
      ). Intersectin (Ese) has five SH3 domains in addition to two EH domains and is involved in the regulation of internalization of the transferrin receptor (
      • Yamabhai M.
      • Hoffman N.G.
      • Hardison N.L.
      • McPherson P.S.
      • Castagnoli L.
      • Cesareni G.
      • Kay B.K.
      ,
      • Sengar A.S.
      • Wang W.
      • Bishay J.
      • Cohen S.
      • Egan S.E.
      ). Pan1 and End3 are Saccharomyces cerevisiae dimeric partners that are necessary for endocytosis of the α-mating factor receptor and for normal organization of the actin cytoskeleton (
      • Benedetti H.
      • Raths S.
      • Crausaz F.
      • Riezman H.
      ,
      • Tang H.Y.
      • Munn A.
      • Cai M.
      ). Thus, the EH domain-containing proteins regulate endocytosis.
      EGF and insulin stimulate the GDP/GTP exchange of Ral through Ras and RalGDS (
      • Kikuchi A.
      • Demo S.D., Ye, Z.-H.
      • Chen Y.-W.
      • Williams L.T.
      ,
      • Kishida S.
      • Koyama S.
      • Matsubara K.
      • Kishida M.
      • Matsuura Y.
      • Kikuchi A.
      ,
      • Wolthuis R.M.F.
      • Zwartkruis F.
      • Moen T.C.
      • Bos J.L.
      ), and the GTP-bound active form of Ral interacts with RalBP1 (
      • Feig L.A.
      • Urano T.
      • Cantor S.
      ). The carboxyl-terminal region of POB1 binds to RalBP1 (
      • Ikeda M.
      • Ishida O.
      • Hinoi T.
      • Kishida S.
      • Kikuchi A.
      ). Because the binding sites of Ral and POB1 on RalBP1 are different, these three proteins form a ternary complex. EGF stimulates tyrosine phosphorylation of POB1 and induces the complex formation between EGF receptor and POB1 (
      • Ikeda M.
      • Ishida O.
      • Hinoi T.
      • Kishida S.
      • Kikuchi A.
      ). The EH domain of POB1 associates with Eps15 and Epsin (
      • Nakashima S.
      • Morinaka K.
      • Koyama S.
      • Ikeda M.
      • Kishida M.
      • Okawa K.
      • Iwamatsu A.
      • Kishida S.
      • Kikuchi A.
      ,
      • Morinaka K.
      • Koyama S.
      • Nakashima S.
      • Hinoi T.
      • Okawa K.
      • Iwamatsu A.
      • Kikuchi A.
      ). Epsin also regulates endocytosis by directly binding to phospholipids (
      • Itoh T.
      • Koshiba S.
      • Kigawa T.
      • Kikuchi A.
      • Yokoyama S.
      • Takenawa T.
      ), α-adaptin (
      • Chen H.
      • Fre S.
      • Slepnev V.I.
      • Capua M.R.
      • Takei K.
      • Butler M.H., Di
      • Fiore P.P.
      • De Camilli P.
      ), and clathrin (
      • Wendland B.
      • Steece K.E.
      • Emr S.D.
      ,
      • Drake M.T.
      • Downs M.A.
      • Traub L.M.
      ). Expression of the EH domain or the carboxyl-terminal region of POB1 inhibits the internalization of EGF and insulin (
      • Nakashima S.
      • Morinaka K.
      • Koyama S.
      • Ikeda M.
      • Kishida M.
      • Okawa K.
      • Iwamatsu A.
      • Kishida S.
      • Kikuchi A.
      ). Therefore, it is conceivable that Ral, RalBP1, and POB1 regulate receptor-mediated endocytosis by transmitting the signal from receptors to Eps15 and Epsin.
      The Arf family of small G proteins is divided into three classes based largely on sequence similarity: class I (Arfs 1–3), class II (Arfs 4 and 5), and class III (Arf6) (
      • Tsuchiya M.
      • Price S.R.
      • Tsai S.C.
      • Moss J.
      • Vaughan M.
      ). By linking GTP binding and hydrolysis, Arfs regulate membrane trafficking at various steps (
      • Boman A.L.
      • Kahn R.A.
      ,
      • Taylor T.C.
      • Kahn R.A.
      • Melancon P.
      ). For instance, Arf6 has been implicated in the regulation of membrane trafficking between the plasma membrane and a specialized endocytic component. Moreover, its function has been linked to cytoskeletal reorganization (
      • Franco M.
      • Peters P.J.
      • Boretto J.
      • van Donselaar E.
      • Neri A.
      • D'Souza-Schorey C.
      • Chavrier P.
      ,
      • Song J.
      • Khachikian Z.
      • Radhakrishna H.
      • Donaldson J.G.
      ). As with other small G proteins, the activity of Arfs is regulated by guanine nucleotide exchange factors and GAPs. It has been shown that ArfGAP is involved in regulating the organization of focal adhesions (
      • Turner C.E.
      ,
      • Turner C.E.
      • West K.A.
      • Brown M.C.
      ). Evidence for a direct link between Arf signaling and focal adhesions came initially from the identification of the ArfGAP protein as a paxillin-binding protein. There are several ArfGAP families. All ArfGAP proteins share homology within the zinc-finger-containing ArfGAP domain and ankyrin repeat region. Among ArfGAP family proteins, PAG3 contains a pleckstrin homology domain in an extended amino terminus and has a proline-rich sequence followed by an SH3 domain at the carboxyl terminus (
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ). PAG3 binds to paxillin and serves as a GAP for Arf6. Overexpression of PAG3 in fibroblasts inhibits cell motility and reduces the paxillin recruitment to focal contacts in a GAP-dependent manner (
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ). These results suggest that PAG3 plays a role in mediating changes in cell motility.
      To find additional functions of POB1, we screened proteins that bind to POB1. Here we report that the proline-rich domain of POB1 interacts with the SH3 domain of PAG2, a PAG3 homolog. Furthermore, we show that the functional interaction of POB1 with PAG2 may regulate cell migration. These results suggest that POB1 and PAG2 link the processes of endocytosis and cell motility.

      DISCUSSION

      In this study, we demonstrated the interaction of POB1 with PAG2. Endogenous PAG2 was detected in the endogenous POB1 immune complex from CHO-IR cells. Sf9-cell-produced GST-POB1 bound to bacterial cell-produced MBP-PAG2-(1002–1132) containing the SH3 domain with aK d value of 13.6 nm. Therefore, it is conceivable that POB1 binds directly to PAG2 under physiological conditions. Furthermore, we demonstrated that the proline-rich motif of POB1 is essential for the binding of POB1 to PAG2. POB1 has three proline-rich motifs, PPTPPPRP345, PPPPALPPRP383, and PPSKPIR428. It is generally thought that the proline-rich motifs bind to several proteins such as profilin and to the EVH1, SH3, and WW domains (
      • Holt M.R.
      • Koffer A.
      ). The core motif that binds to the SH3 domain is PXXP, and this motif is further classified into class I and class II. The class I motif is (R/K)XXPXXP, which binds to the SH3 domains of Src, Abl, Fyn, and Lyn. The class II motif is PXXPX(R/K), which binds to the SH3 domains of Grb2, Nck, and Crk. All of the proline-rich motifs of POB1 are class II of the SH3-domain binding motifs. Because substitution of two proline residues with alanine in the third motif of POB1 impaired its binding to PAG2, the third proline motif is essential for the binding to PAG2. These results suggest that the proline-rich motifs of POB1 interact with the SH3 domain of PAG2. It has been shown that the SH3 domain of PAG3/PAPα binds to Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of PAPα (
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ,
      • Andreev J.
      • Simon J.P.
      • Sabatini D.D.
      • Kam J.
      • Plowman G.
      • Randazzo P.A.
      • Schlessinger J.
      ). Because the SH3 domain of PAG2 shares 76% identity with that of PAG3, PAG2 may interact with Pyk2. It remains to be clarified whether POB1 affects the interaction of PAG2 with Pyk2. Previously we showed that among the SH3-domain-containing proteins, Grb2 but not Nck and Crk binds to POB1 (
      • Ikeda M.
      • Ishida O.
      • Hinoi T.
      • Kishida S.
      • Kikuchi A.
      ). Because Grb2 bound to both POB1 and POB1(PA) (data not shown), it seems that the third proline-rich motif of POB1 is not essential for its binding to Grb2, suggesting that PAG2 and Grb2 bind to different sites of POB1.
      Several lines of evidence indicate that ArfGAP family members, including GIT, PAG3/PAPα, and ASAP1/DEF-1, regulate actin cytoskeletal dynamics (
      • Turner C.E.
      • West K.A.
      • Brown M.C.
      ). PAG3 interacts with paxillin, which acts as an adaptor molecule in integrin signaling and is localized to focal contacts (
      • Turner C.E.
      ,
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ). PAG3 is diffusely distributed in the cytoplasm in premature monocytes but becomes localized at cell periphery in mature monocytes (
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ). However, PAG3 does not accumulate at focal contacts, suggesting that PAG3 is not an integrin assembly protein. Overexpression of PAG3 in COS-7 and U937 cells causes a loss of the paxillin recruitment to focal adhesions and inhibits cell motility in a GAP-dependent manner. Overexpression of PAG2 also impaired cell migratory activities and inhibited paxillin recruitment to focal contacts. This does not always reflect that PAG2 negatively regulates cell migration because overexpression of PAG2 may interfere with the functions of proteins that are involved in the cell migration through recruitment of the binding partners even though PAG2 is a positive regulator.
      The amino-terminal region of PAG2 containing the ArfGAP domain, but not the carboxyl-terminal region containing the binding sites of paxillin and POB1, inhibited cell migration. Taken together with the observations that the activities of Arfs are involved in the focal adhesion recruitment of paxillin (
      • Kondo A.
      • Hashimoto S.
      • Yano H.
      • Nagayama K.
      • Mazaki Y.
      • Sabe H.
      ,
      • Norman J.
      • Jones D.
      • Barry S.
      • Holt M.
      • Cockcroft S.
      • Critchley D.
      ), it is conceivable that the ArfGAP activity is essential for these activities of PAG2, but we do not know the physiological roles of the paxillin-binding activity of PAG2 for them. We also showed that POB1, but not POB1(PA), restores cell motility and the paxillin recruitment to focal contacts, which are inhibited by PAG2. Moreover, POB1 could not rescue the inhibition by the amino-terminal region of PAG2 that lacks the POB1-binding site. Therefore, the interaction of POB1 with PAG2 may regulate the paxillin recruitment to focal contacts, but we do not know the mechanism at present. One possibility might be that POB1 participates in the recruitment of PAG2 to proper subcellular areas in which PAG2 may act as a GAP for Arfs, resulting in the regulation of the subcellular positioning of paxillin. It has been proposed that primer proteins including the coatmer and the GTP-bound form of Arf at the membranes of the endoplasmic reticulum and the Golgi apparatus influence the catalytic activity of ArfGAP1 (
      • Springer S.
      • Spang A.
      • Schekman R.
      , ). Therefore, complex formation between paxillin, PAG2, and POB1 at certain subcellular areas may constitute a signal necessary for the onset or the enhancement of the catalytic GAP activity of PAG2 toward the GTP-bound form of Arfs.
      Cell locomotion is driven by protrusive activity at the leading edge of the cell where continuous remodeling of actin cytoskelton and adhesive contacts is required (
      • Bretscher M.S.
      ). Endocytosed membrane is reinserted at the leading edge of migrating cells, extending the front of the cell forward. For instance, recycling transferrin receptors and low density lipoprotein receptors are distributed to the cell front of migrating fibroblasts and Rac-induced ruffles (
      • Hopkins C.R.
      • Gibson A.
      • Shipman M.
      • Strickland D.K.
      • Trowbridge I.S.
      ,
      • Bretscher M.S.
      • Aguado-Velasco C.
      ). Therefore, it is likely that the random reinsertion of internalized membranes at the surface of a resting cell is redirected to the site of protrusion when migration is induced by mitogenic stimuli. Arf6 is implicated in the regulation of membrane trafficking between the recycling endosomal compartment and the plasma membrane, based on the specific localization of Arf6 in these compartments and the effects of its overexpression on transferrin uptake and recycling to the cell surface (
      • D'Souza-Schorey C., Li, G.
      • Colombo M.I.
      • Stahl P.D.
      ,
      • Peters P.J.
      • Hsu V.W.
      • Ooi C.E.
      • Finazzi D.
      • Teal S.B.
      • Oorschot V.
      • Donaldson J.G.
      • Klausner R.D.
      ). Arf6 co-localizes with Rac1, which is involved in the formation of actin-rich ruffles and lamellipodia, at the plasma membrane and on recycling endosomes (
      • Radhakrishna H., Al-
      • Awar O.
      • Khachikian Z.
      • Donaldson J.G.
      ). Moreover, the ArfGAP family proteins interact with proteins involved in both cell adhesion and actin organization (
      • Turner C.E.
      • West K.A.
      • Brown M.C.
      ). Therefore, it has been speculated that ArfGAP is involved in the regulation of Arf-mediated membrane recycling and protrusion during cell locomotion.
      We have demonstrated that small G protein Ral and its downstream molecules, RalBP1 and POB1, are involved in receptor-mediated endocytosis of EGF and insulin (
      • Nakashima S.
      • Morinaka K.
      • Koyama S.
      • Ikeda M.
      • Kishida M.
      • Okawa K.
      • Iwamatsu A.
      • Kishida S.
      • Kikuchi A.
      ). Furthermore, we have found that Eps15 and Epsin bind directly to the EH domain of POB1 (
      • Nakashima S.
      • Morinaka K.
      • Koyama S.
      • Ikeda M.
      • Kishida M.
      • Okawa K.
      • Iwamatsu A.
      • Kishida S.
      • Kikuchi A.
      ,
      • Morinaka K.
      • Koyama S.
      • Nakashima S.
      • Hinoi T.
      • Okawa K.
      • Iwamatsu A.
      • Kikuchi A.
      ). These results suggest that the signaling from Ral to Eps15 and Epsin through RalBP1 and POB1 regulates receptor-mediated endocytosis. Because Eps15 and Epsin are core proteins that regulate endocytosis, POB1 may be able to link endocytosis and cell migration. The binding sites of POB1 for Epsin, RalBP1, and PAG2 are different. Taken together with the observations that Ral regulates both actin cytoskeletal remodeling and vesicle transport (
      • Nakashima S.
      • Morinaka K.
      • Koyama S.
      • Ikeda M.
      • Kishida M.
      • Okawa K.
      • Iwamatsu A.
      • Kishida S.
      • Kikuchi A.
      ,
      • Suzuki J.
      • Yamazaki Y., Li, G.
      • Kaziro Y.
      • Koide H.
      ,
      • Moskalenko S.
      • Henry D.O.
      • Rosse C.
      • Mirey G.
      • Camonis J.H.
      • White M.A.
      ,
      • Sugihara K.
      • Asano S.
      • Tanaka K.
      • Iwamatsu A.
      • Okawa K.
      • Ohta Y.
      ), it is intriguing to speculate that POB1 may function as a scaffold protein in that it interacts with proteins involved in endocytosis and migration to create a multi-protein complex. Further analysis would be necessary to understand how these complex interactions are temporally and spacially coordinated during cell migration.

      Acknowledgments

      We thank Drs. Y. Matsuura and Q. Hu for donating reagents.

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