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

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.

[1]  M. A. Downs,et al.  Epsin Binds to Clathrin by Associating Directly with the Clathrin-terminal Domain , 2000, The Journal of Biological Chemistry.

[2]  S. Demo,et al.  ralGDS family members interact with the effector loop of ras p21 , 1994, Molecular and cellular biology.

[3]  H. Radhakrishna,et al.  Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading. , 1998, Journal of cell science.

[4]  P. Pelicci,et al.  eps15 and eps15R are essential components of the endocytic pathway. , 1997, Cancer research.

[5]  T. Kigawa,et al.  Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. , 2001, Science.

[6]  A. Iwamatsu,et al.  The exocyst complex binds the small GTPase RalA to mediate filopodia formation , 2002, Nature Cell Biology.

[7]  M. Bretscher,et al.  EGF induces recycling membrane to form ruffles , 1998, Current Biology.

[8]  D. Görlich,et al.  Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPα but not importin α , 1999 .

[9]  R. Wolthuis,et al.  Ras-dependent activation of the small GTPase Ral , 1998, Current Biology.

[10]  K. Vuori,et al.  CAS/Crk Coupling Serves as a “Molecular Switch” for Induction of Cell Migration , 1998, The Journal of cell biology.

[11]  P. Pelicci,et al.  Mapping of the molecular determinants involved in the interaction between eps15 and AP-2. , 1997, Cancer research.

[12]  Pier Paolo Di Fiore,et al.  Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis , 1998, Nature.

[13]  L. Castagnoli,et al.  Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains* , 1998, The Journal of Biological Chemistry.

[14]  D. Strickland,et al.  In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella , 1994, The Journal of cell biology.

[15]  M. Bretscher.  Getting Membrane Flow and the Cytoskeleton to Cooperate in Moving Cells , 1996, Cell.

[16]  S. Cantor,et al.  Evidence for a Ras/Ral signaling cascade. , 1996, Trends in biochemical sciences.

[17]  R. Klausner,et al.  Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments , 1995, The Journal of cell biology.

[18]  M. Brown,et al.  Paxillin-ARF GAP signaling and the cytoskeleton. , 2001, Current opinion in cell biology.

[19]  M R Holt,et al.  Cell motility: proline-rich proteins promote protrusions. , 2001, Trends in cell biology.

[20]  R. Schekman,et al.  A Primer on Vesicle Budding , 1999, Cell.

[21]  A. Munn,et al.  EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae , 1997, Molecular and cellular biology.

[22]  R. Kahn,et al.  Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-golgi transport , 1992, Cell.

[23]  S. Schmid,et al.  AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis , 1998, The Journal of cell biology.

[24]  A. Kikuchi,et al.  Ras-interacting domain of Ral GDP dissociation stimulator like (RGL) reverses v-Ras-induced transformation and Raf-1 activation in NIH3T3 cells. , 1996, Cancer research.

[25]  Li Guang,et al.  Involvement of Ras and Ral in Chemotactic Migration of Skeletal Myoblasts , 2000, Molecular and Cellular Biology.

[26]  B. Kay,et al.  The EH network. , 1999, Experimental cell research.

[27]  A. Iwamatsu,et al.  Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis , 1999, Oncogene.

[28]  K. Nagayama,et al.  A new paxillin-binding protein, PAG3/Papalpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. , 2000, Molecular biology of the cell.

[29]  R. Kahn,et al.  Arf proteins: the membrane traffic police? , 1995, Trends in biochemical sciences.

[30]  L. Minichiello,et al.  eps15, a novel tyrosine kinase substrate, exhibits transforming activity , 1993, Molecular and cellular biology.

[31]  A. Tsubouchi,et al.  An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization. , 2001, Molecular biology of the cell.

[32]  C. Rossé,et al.  The exocyst is a Ral effector complex , 2002, Nature Cell Biology.

[33]  M. Roth,et al.  Snapshots of ARF1 Implications for Mechanisms of Activation and Inactivation , 1999, Cell.

[34]  S. Barry,et al.  ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts , 1998, The Journal of cell biology.

[35]  T. Urano,et al.  An Eps Homology (EH) Domain Protein That Binds to the Ral-GTPase Target, RalBP1* , 1997, The Journal of Biological Chemistry.

[36]  C. Turner.  Paxillin and focal adhesion signalling , 2000, Nature Cell Biology.

[37]  A. Kikuchi,et al.  The post‐translational modifications of Ral and Rac1 are important for the action of Ral‐binding protein 1, a putative effector protein of Ral , 1997, FEBS letters.

[38]  T. Hinoi,et al.  Identification and Characterization of a Novel Protein Interacting with Ral-binding Protein 1, a Putative Effector Protein of Ral* , 1998, The Journal of Biological Chemistry.

[39]  Jonathan A. Cooper,et al.  ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src , 1998, Molecular and Cellular Biology.

[40]  S. Egan,et al.  The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15 , 1999, The EMBO journal.

[41]  H. Riezman,et al.  The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast , 1994, Molecular biology of the cell.

[42]  K. Morinaka,et al.  Regulation of Complex Formation of POB1/Epsin/Adaptor Protein Complex 2 by Mitotic Phosphorylation* , 2000, The Journal of Biological Chemistry.

[43]  P. Pelicci,et al.  Eps15R Is a Tyrosine Kinase Substrate with Characteristics of a Docking Protein Possibly Involved in Coated Pits-mediated Internalization* , 1998, The Journal of Biological Chemistry.

[44]  E. V. van Donselaar,et al.  EFA6, a sec7 domain‐containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization , 1999, The EMBO journal.

[45]  T. Kataoka,et al.  Synergistic activation of c-fos promoter activity by Raf and Ral GDP dissociation stimulator , 1997, Oncogene.

[46]  P. Pelicci,et al.  A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[47]  H. Radhakrishna,et al.  ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. , 1999, Journal of cell science.

[48]  Y. Matsuura,et al.  Colocalization of Ras and Ral on the membrane is required for Ras-dependent Ral activation through Ral GDP dissociation stimulator , 1997, Oncogene.

[49]  C. D’Souza-Schorey,et al.  A regulatory role for ARF6 in receptor-mediated endocytosis , 1995, Science.

[50]  A. Iwamatsu,et al.  Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors , 1999, The EMBO journal.

[51]  J. Schlessinger,et al.  Identification of a New Pyk2 Target Protein with Arf-GAP Activity , 1999, Molecular and Cellular Biology.

[52]  J. Moss,et al.  Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells. , 1991, The Journal of biological chemistry.