G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
BAI1-associated protein 2
G00000012 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000141742 (Vega human gene)
ENSG00000175866 (Ensembl human gene)
10458 (Entrez Gene)
471 (G2Cdb plasticity & disease)
BAIAP2 (GeneCards)
605475 (OMIM)
Marker Symbol
Protein Sequence
Q9UQB8 (UniProt)

Synonyms (1)

  • BAP2

Literature (42)

Pubmed - other

  • Regulation of IRSp53-dependent filopodial dynamics by antagonism between 14-3-3 binding and SH3-mediated localization.

    Robens JM, Yeow-Fong L, Ng E, Hall C and Manser E

    RGS Group, Institute of Medical Biology,Singapore 138673, Singapore.

    Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.

    Molecular and cellular biology 2010;30;3;829-44

  • Case-control study of six genes asymmetrically expressed in the two cerebral hemispheres: association of BAIAP2 with attention-deficit/hyperactivity disorder.

    Ribasés M, Bosch R, Hervás A, Ramos-Quiroga JA, Sánchez-Mora C, Bielsa A, Gastaminza X, Guijarro-Domingo S, Nogueira M, Gómez-Barros N, Kreiker S, Gross-Lesch S, Jacob CP, Lesch KP, Reif A, Johansson S, Plessen KJ, Knappskog PM, Haavik J, Estivill X, Casas M, Bayés M and Cormand B

    Department of Psychiatry, Hospital Universitari Vall d'Hebron, Barcelona, Catalonia, Spain.

    Background: Attention-deficit/hyperactivity disorder (ADHD) is a childhood-onset neuropsychiatric disease that persists into adulthood in at least 30% of patients. There is evidence suggesting that abnormal left-right brain asymmetries in ADHD patients may be involved in a variety of ADHD-related cognitive processes, including sustained attention, working memory, response inhibition and planning. Although mechanisms underlying cerebral lateralization are unknown, left-right cortical asymmetry has been associated with transcriptional asymmetry at embryonic stages and several genes differentially expressed between hemispheres have been identified.

    Methods: We selected six functional candidate genes showing at least 1.9-fold differential expression between hemispheres (BAIAP2, DAPPER1, LMO4, NEUROD6, ATP2B3, and ID2) and performed a case-control association study in an initial Spanish sample of 587 ADHD patients (270 adults and 317 children) and 587 control subjects.

    Results: The single- and multiple-marker analysis provided evidence for a contribution of BAIAP2 to adulthood ADHD (p = .0026 and p = .0016, respectively). We thus tested BAIAP2 for replication in two independent adult samples from Germany (639 ADHD patients and 612 control subjects) and Norway (417 ADHD cases and 469 control subjects). While no significant results were observed in the Norwegian sample, we replicated the initial association between BAIAP2 and adulthood ADHD in the German population (p = .0062).

    Conclusions: Our results support the participation of BAIAP2 in the continuity of ADHD across life span, at least in some of the populations analyzed, and suggest that genetic factors potentially influencing abnormal cerebral lateralization may be involved in this disorder.

    Biological psychiatry 2009;66;10;926-34

  • Interaction between enterohemorrhagic Escherichia coli O157:H7 EspFu and IRSp53 induces dynamic membrane remodeling in epithelial cells.

    Morita-Ishihara T, Terajima J, Watanabe H and Izumiya H

    Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 induces the formation of filamentous, actin-rich, pedestal-shaped structures beneath bacterial cells that have attached to intestinal epithelial cells. Pedestal formation requires the translocation of EHEC O157:H7 type III effectors. One of these type III effectors, EspFu, consists of an N-terminal signal sequence, which is necessary for the translocation of EspFu into the host cell through a type III secretion system, and almost identical proline-rich repeats (PRRs), which control actin rearrangement and increase the efficiency of actin assembly in the host cell. In this study, we report that insulin receptor tyrosine kinase substrate p53 (IRSp53) in the host cell acts as a binding partner for EspFu. Co-immunoprecipitation and fluorescence microscopy showed specific interactions between EspFu and IRSp53 as well as their co-localization in epithelial cells. Additionally, we demonstrated that the association between EspFu and IRSp53 induces dynamic membrane remodeling in epithelial cells.

    Japanese journal of infectious diseases 2009;62;5;351-5

  • SPIN90-IRSp53 complex participates in Rac-induced membrane ruffling.

    Teodorof C, Bae JI, Kim SM, Oh HJ, Kang YS, Choi J, Chun JS and Song WK

    Bio Imaging Research Center, Cell Dynamics Research Center, Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, South Korea.

    SPIN90 is a key regulator of actin cytoskeletal organization. Using the BioGRID(beta) database (General Repository for Interaction Datasets), we identified IRSp53 as a binding partner of SPIN90, and confirmed the in vivo formation of a SPIN90-IRSp53 complex mediated through direct association of the proline-rich domain (PRD) of SPIN90 with the SH3 domain of IRSp53. SPIN90 and IRSp53 positively cooperated to mediate Rac activation, and co-expression of SPIN90 and IRSp53 in COS-7 cells led to the complex formation of SPIN90-IRSp53 in the leading edge of cells. PDGF treatment induced strong colocalization of SPIN90 and IRSp53 at membrane protrusions. Within such PDGF-induced protrusions, knockdown of SPIN90 protein using siRNA significantly reduced lamellipodia-like protrusions as well as localization of IRSp53 at those sites. Finally, competitive inhibition of SPIN90-IRSp53 binding by SPIN90 PRD dramatically reduced ruffle formation, further suggesting that SPIN90 plays a key role in the formation of the membrane protrusions associated with cell motility.

    Experimental cell research 2009;315;14;2410-9

  • IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.

    Weiss SM, Ladwein M, Schmidt D, Ehinger J, Lommel S, Städing K, Beutling U, Disanza A, Frank R, Jänsch L, Scita G, Gunzer F, Rottner K and Stradal TE

    Signalling and Motility Group, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany.

    Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation.

    Cell host & microbe 2009;5;3;244-58

  • Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53.

    Kim MH, Choi J, Yang J, Chung W, Kim JH, Paik SK, Kim K, Han S, Won H, Bae YS, Cho SH, Seo J, Bae YC, Choi SY and Kim E

    National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    IRSp53 is an adaptor protein that acts downstream of Rac and Cdc42 small GTPases and is implicated in the regulation of membrane deformation and actin filament assembly. In neurons, IRSp53 is an abundant postsynaptic protein and regulates actin-rich dendritic spines; however, its in vivo functions have not been explored. We characterized transgenic mice deficient of IRSp53 expression. Unexpectedly, IRSp53(-/-) neurons do not show significant changes in the density and ultrastructural morphologies of dendritic spines. Instead, IRSp53(-/-) neurons exhibit reduced AMPA/NMDA ratio of excitatory synaptic transmission and a selective increase in NMDA but not AMPA receptor-mediated transmission. IRSp53(-/-) hippocampal slices show a markedly enhanced long-term potentiation (LTP) with no changes in long-term depression. LTP-inducing theta burst stimulation enhances NMDA receptor-mediated transmission. Spatial learning and novel object recognition are impaired in IRSp53(-/-) mice. These results suggest that IRSp53 is involved in the regulation of NMDA receptor-mediated excitatory synaptic transmission, LTP, and learning and memory behaviors.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;5;1586-95

  • LIN7 mediates the recruitment of IRSp53 to tight junctions.

    Massari S, Perego C, Padovano V, D'Amico A, Raimondi A, Francolini M and Pietrini G

    Department of Medical Pharmacology, Institute of Neuroscience-Consiglio Nazionale delle Richerche, University of Milan, 20129 Milan, Italy.

    In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (DeltaL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.

    Traffic (Copenhagen, Denmark) 2009;10;2;246-57

  • Kank attenuates actin remodeling by preventing interaction between IRSp53 and Rac1.

    Roy BC, Kakinuma N and Kiyama R

    Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.

    In this study, insulin receptor substrate (IRS) p53 is identified as a binding partner for Kank, a kidney ankyrin repeat-containing protein that functions to suppress cell proliferation and regulate the actin cytoskeleton. Kank specifically inhibits the binding of IRSp53 with active Rac1 (Rac1(G12V)) but not Cdc42 (cdc42(G12V)) and thus inhibits the IRSp53-dependent development of lamellipodia without affecting the formation of filopodia. Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect. Moreover, insulin-induced membrane ruffling is inhibited by overexpression of Kank. Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth. Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

    The Journal of cell biology 2009;184;2;253-67

  • Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53.

    Heung MY, Visegrady B, Fütterer K and Machesky LM

    School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

    The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.

    Funded by: Medical Research Council: G117/569

    European journal of cell biology 2008;87;8-9;699-708

  • IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions.

    Scita G, Confalonieri S, Lappalainen P and Suetsugu S

    IFOM (FIRC Institute for Molecular Oncology) - Foundation, Via Adamello 16, 20139 Milan, Italy; School of Medicine, University of Milan, 20122 Milan, Italy.

    A tight spatiotemporal coordination of the machineries controlling membrane bending and trafficking, and actin dynamics is crucial for the generation of cellular protrusions. Proteins that are simultaneously capable of regulating actin dynamics and sensing or inducing membrane curvature are predicted to have a prominent role. A prototypical example of this type of proteins is the insulin receptor tyrosine kinase substrate of 53kDa, the founding member of a recently discovered family of proteins, including missing-in-metastasis and ABBA (actin-bundling protein with BAIAP2 homology). Structural, biochemical and cell biological experiments support the unique role of this family as transducers of signalling, linking the protruding membrane to the underlying actin cytoskeleton.

    Trends in cell biology 2008;18;2;52-60

  • Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism.

    Mattila PK, Pykäläinen A, Saarikangas J, Paavilainen VO, Vihinen H, Jokitalo E and Lappalainen P

    Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.

    The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P(2)-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P(2)-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin-bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis.

    The Journal of cell biology 2007;176;7;953-64

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex.

    Disanza A, Mantoani S, Hertzog M, Gerboth S, Frittoli E, Steffen A, Berhoerster K, Kreienkamp HJ, Milanesi F, Di Fiore PP, Ciliberto A, Stradal TE and Scita G

    Istituto FIRC di Oncologia Molecolare (IFOM), Via Adamello 16, 20139, Milan, Italy.

    Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.

    Nature cell biology 2006;8;12;1337-47

  • The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation.

    Suetsugu S, Murayama K, Sakamoto A, Hanawa-Suetsugu K, Seto A, Oikawa T, Mishima C, Shirouzu M, Takenawa T and Yokoyama S

    Department of Biochemistry, Institute of Medical Science, the University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, USA.

    The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.

    The Journal of biological chemistry 2006;281;46;35347-58

  • Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP(3), and Rac.

    Suetsugu S, Kurisu S, Oikawa T, Yamazaki D, Oda A and Takenawa T

    Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

    WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).

    The Journal of cell biology 2006;173;4;571-85

  • A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.

    Lim J, Hao T, Shaw C, Patel AJ, Szabó G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, Barabási AL, Vidal M and Zoghbi HY

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.

    Funded by: NICHD NIH HHS: HD24064; NINDS NIH HHS: NS27699

    Cell 2006;125;4;801-14

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Regulation of dendritic spine morphogenesis by insulin receptor substrate 53, a downstream effector of Rac1 and Cdc42 small GTPases.

    Choi J, Ko J, Racz B, Burette A, Lee JR, Kim S, Na M, Lee HW, Kim K, Weinberg RJ and Kim E

    National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac1/Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Rac1/Cdc42 to downstream effectors for actin regulation in non-neural cells. Here, we report that IRSp53 interacts with two specific members of the PSD-95 family, PSD-95 and chapsyn-110/PSD-93, in brain. An IRSp53 mutant lacking the C-terminal PSD-95-binding motif shows significant loss of synaptic localization in cultured neurons. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines but does not affect their length or width. Conversely, short-interfering RNA-mediated knock-down of IRSp53 reduces the density, length, and width of spines. In addition, the density and size of spines are decreased by a dominant-negative IRSp53 with a point mutation in the Src homology 3 (SH3) domain and a dominant-negative proline-rich region of WAVE2 (Wiskott-Aldrich syndrome protein family Verprolin-homologous protein), a downstream effector of IRSp53 that binds to the SH3 domain of IRSp53. These results suggest that PSD-95 interaction is an important determinant of synaptic IRSp53 localization and that the SH3 domain of IRSp53 links activated Rac1/Cdc42 to downstream effectors for the regulation of spine morphogenesis.

    Funded by: NINDS NIH HHS: NS-39444

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;4;869-79

  • Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53.

    Millard TH, Bompard G, Heung MY, Dafforn TR, Scott DJ, Machesky LM and Fütterer K

    School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, UK.

    The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.

    Funded by: Medical Research Council: MRC_G117/379

    The EMBO journal 2005;24;2;240-50

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness.

    Funato Y, Terabayashi T, Suenaga N, Seiki M, Takenawa T and Miki H

    Division of Cancer Genomics, Biochemistry, and Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    IRSp53 has been characterized as an adaptor protein that links Rho-family small GTPases, such as Rac, to reorganization of the actin cytoskeleton. Here, we search for other binding partners for the IRSp53 SH3 domain and identify Eps8 as the major binding protein in fibroblasts and various cancer cell lines. Eps8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with Abi-1 and Sos-1, which seems essential for ruffling formation induced by oncogenic Ras. We confirm the IRSp53/Eps8 complex formation in vivo and the direct association between Eps8 NH(2)-terminal proline-rich sequence and IRSp53 SH3 domain. This complex synergistically activates Rac by reinforcing the formation of the Eps8/Abi-1/Sos-1 Rac-guanine nucleotide exchange factor complex, which mediates positive regulation of Rac activity. In addition, IRSp53/Eps8 complex formation as determined by fluorescent resonance energy transfer analysis, occurs at the leading edge of motile cells, and the motility and invasiveness of HT1080 fibrosarcoma cells are suppressed by inhibiting complex formation. These findings implicate the importance of the IRSp53/Eps8 complex in Rac activation and metastatic behavior of the malignant tumor cells.

    Cancer research 2004;64;15;5237-44

  • A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein.

    Yamagishi A, Masuda M, Ohki T, Onishi H and Mochizuki N

    Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan.

    Insulin receptor tyrosine kinase substrate p53 (IRSp53) has been identified as an SH3 domain-containing adaptor that links Rac1 with a Wiskott-Aldrich syndrome family verprolin-homologous protein 2 (WAVE2) to induce lamellipodia or Cdc42 with Mena to induce filopodia. The recruitment of these SH3-binding partners by IRSp53 is thought to be crucial for F-actin rearrangements. Here, we show that the N-terminal predicted helical stretch of 250 amino acids of IRSp53 is an evolutionarily conserved F-actin bundling domain involved in filopodium formation. Five proteins including IRSp53 and missing in metastasis (MIM) protein share this unique domain and are highly conserved in vertebrates. We named the conserved domain IRSp53/MIM homology domain (IMD). The IMD has domain relatives in invertebrates but does not show obvious homology to any known actin interacting proteins. The IMD alone, derived from either IRSp53 or MIM, induced filopodia in HeLa cells and the formation of tightly packed parallel F-actin bundles in vitro. These results suggest that IRSp53 and MIM belong to a novel actin bundling protein family. Furthermore, we found that filopodium-inducing IMD activity in the full-length IRSp53 was regulated by active Cdc42 and Rac1. The SH3 domain was not necessary for IMD-induced filopodium formation. Our results indicate that IRSp53, when activated by small GTPases, participates in F-actin reorganization not only in an SH3-dependent manner but also in a manner dependent on the activity of the IMD.

    The Journal of biological chemistry 2004;279;15;14929-36

  • Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region.

    Lehner B, Semple JI, Brown SE, Counsell D, Campbell RD and Sanderson CM

    Functional Genomics Group, MRC Rosalind Franklin Centre for Genomics Research, Hinxton, Cambridge, United Kingdom.

    High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.

    Genomics 2004;83;1;153-67

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • MALS is a binding partner of IRSp53 at cell-cell contacts.

    Hori K, Konno D, Maruoka H and Sobue K

    Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka, Japan.

    Insulin receptor substrate p53 (IRSp53) is a key player in cytoskeletal dynamics, interacting with the actin modulators WAVE2 and Mena. Here, we identified a PDZ protein, MALS, as an IRSp53-interacting protein using a yeast two-hybrid screen. A pull-down assay showed that IRSp53 and MALS interact through the PDZ domain of MALS and the C-terminal PDZ-binding sequence of IRSp53. Their interaction in MDCK cells was also demonstrated by co-immunoprecipitation. Immunocytochemistry showed the colocalization of IRSp53 and MALS at cell-cell contacts. Cytochalasin D induced the redistribution of both proteins to the cytosol. Thus, MALS is a partner of IRSp53 anchoring the actin-based membrane cytoskeleton at cell-cell contacts.

    FEBS letters 2003;554;1-2;30-4

  • Novel espin actin-bundling proteins are localized to Purkinje cell dendritic spines and bind the Src homology 3 adapter protein insulin receptor substrate p53.

    Sekerková G, Loomis PA, Changyaleket B, Zheng L, Eytan R, Chen B, Mugnaini E and Bartles JR

    Department of Cell and Molecular Biology, Feinberg School of Medicine and the Institute for Neuroscience, Northwestern University, Chicago, Illinois 60611, USA.

    We identified a group of actin-binding-bundling proteins that are expressed in cerebellar Purkinje cells (PCs) but are not detected in other neurons of the CNS. These proteins are novel isoforms of the actin-bundling protein espin that arise through the use of a unique site for transcriptional initiation and differential splicing. Light and electron microscopic localization studies demonstrated that these espin isoforms are enriched in the dendritic spines of PCs. They were detected in the head and neck and in association with the postsynaptic density (PSD) of dendritic spines in synaptic contact with parallel or climbing fibers. They were also highly enriched in PSD fractions isolated from cerebellum. The PC espins efficiently bound and bundled actin filaments in vitro, and these activities were not inhibited by Ca2+. When expressed in transfected neuronal cell lines, the PC espins colocalized with actin filaments and elicited the formation of coarse cytoplasmic actin bundles. The insulin receptor substrate p53 (IRSp53), an Src homology 3 (SH3) adapter protein and regulator of the actin cytoskeleton, was identified as an espin-binding protein in yeast two-hybrid screens. Cotransfection studies and pull-down assays showed that this interaction was direct and required the N-terminal proline-rich peptide of the PC espins. Thus, the PC espins exhibit the properties of modular actin-bundling proteins with the potential to influence the organization and dynamics of the actin cytoskeleton in PC dendritic spines and to participate in multiprotein complexes involving SH3 domain-containing proteins, such as IRSp53.

    Funded by: NICHD NIH HHS: HD01210, K02 HD001210, K02 HD001210-05; NIDCD NIH HHS: DC04314, R01 DC004314, R01 DC004314-03

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;4;1310-9

  • Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein.

    Miyahara A, Okamura-Oho Y, Miyashita T, Hoshika A and Yamada M

    Department of Genetics, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan.

    Insulin receptor tyrosine kinase substrate of 53-kDa protein (IRSp53) is now known to be a key factor in cytoskeleton reorganization. The human IRSp53 was identified as a binding partner with DRPLA protein, a product of the gene responsible for a neurodegenerative disorder, dentatorubral pallidoluysian atrophy, as well as a binding partner with brain-specific angiogenesis inhibitor 1. Previous studies identified at least four isoforms (L-, M-, S- and T-forms) in human, where 511 amino acid residues from the N-terminus were identical, followed by unique sequences of 9-41 amino acid residues. As each isoform had a distinct function, the unique sequences at the C-terminus had a vital role in its function. Here we report that these isoforms were indeed generated by alternative splicing, which was established by experimental and computational studies on human and rodent genomes. Previous biochemical reports suggested that rodents may lack one of the isoforms (L-form). This study solved this issue, as a nucleotide substitution occurred at a splice donor site followed by a large deletion in the rodent genome compared with human, which made the generation of the L-form impossible. This study also revealed overlapping of the IRSp53 and AATK genes coded for by complementary strands.

    Journal of human genetics 2003;48;8;410-4

  • The insulin receptor substrate IRSp53 links postsynaptic shank1 to the small G-protein cdc42.

    Soltau M, Richter D and Kreienkamp HJ

    Institut für Zellbiochemie und klinische Neurobiologie, Universitätskrankenhaus Eppendorf, Hamburg, Germany.

    The multidomain shank/ProSAP/SSTRIP proteins are major scaffold proteins in glutamatergic synapses in the mammalian brain; expression of shank1/SSTRIP in hippocampal neurons induces morphological changes in dendritic spines, suggesting that shank1 is involved in synapse formation and activity-dependent changes of synaptic structure. Using part of the proline-rich region of shank1 in a yeast two hybrid screen, we identified the insulin receptor substrate IRSp53 as an interaction partner. Overlay assays verified a strong interaction between a proline-rich sequence (residues 911-940) in shank1 and the SH3 domain of IRSp53. When coexpressed in HEK cells, shank1 colocalizes with IRSp53 in intracellular structures, preventing targeting of IRSp53 to filopodia which are induced by IRSp53 expression in the absence of shank1. IRSp53 also binds to the activated form of the small G-protein cdc42. Interestingly, IRSp53 coprecipitates with shank1 from transfected HEK cells in a small G-protein-regulated manner. Thus, IRSp53 constitutes a cdc42-regulated ligand for shank1 which may provide a molecular basis for small G-protein mediated effects on the structure of the postsynaptic complex.

    Molecular and cellular neurosciences 2002;21;4;575-83

  • WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac.

    Miki H and Takenawa T

    Division of Cancer Genomics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    We previously reported that IRSp53 binds both Rac and WAVE2, inducing formation of Rac/IRSp53/WAVE2 complex that is important for membrane ruffling. However, recent reports noted a specific interaction between IRSp53 and Cdc42 but not Rac, which led us to re-examine the binding of IRSp53 to Rac. Immunoprecipitation analysis and pull-down assay reveal that full-length IRSp53 binds Rac much less efficiently than the N-terminal fragment, which may be caused by intramolecular interaction. Interestingly, the intramolecular interaction is interrupted by the binding of WAVE2 and full-length IRSp53 associates with Rac in the presence of WAVE2. We also report that IRSp53 induces spreading and neurite formation of N1E-115 cells, which presumably reflect functional cooperation with Rac.

    Biochemical and biophysical research communications 2002;293;1;93-9

  • Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.

    Krugmann S, Jordens I, Gevaert K, Driessens M, Vandekerckhove J and Hall A

    MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, CRC Oncogene and Signal Transduction Group, University College London, Gower Street, London WC1E 6BT, United Kingdom.

    Background: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear.

    Results: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation.

    Conclusion: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.

    Current biology : CB 2001;11;21;1645-55

  • Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.

    Govind S, Kozma R, Monfries C, Lim L and Ahmed S

    Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.

    Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

    The Journal of cell biology 2001;152;3;579-94

  • IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling.

    Miki H, Yamaguchi H, Suetsugu S and Takenawa T

    Department of Biochemistry, Institute of Medical Science, University of Tokyo, and CREST, Japan Science and Technology Corporation.

    Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42 (refs 7, 8), which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. Here we demonstrate that IRSp53, a substrate for insulin receptor with unknown function, is the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, we found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex.

    Nature 2000;408;6813;732-5

  • Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2.

    Fujiwara T, Mammoto A, Kim Y and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/ Faculty of Medicine, Suita, 565-0871, Japan.

    mDia1 is a downstream effector of Rho small G protein that is implicated in stress fiber formation and cytokinesis. We isolated an mDia1-binding protein and identified it to be IRSp53/BAIAP2. IRSp53 and BAIAP2 have independently been isolated as a 58/53-kDa protein tyrosine phosphorylated in response to insulin and a BAI1-binding protein, respectively. BAI1 is a brain-specific seven-span transmembrane protein capable of inhibiting angiogenesis. The proline-rich formin homology 1 domain of mDia1 bound the Src homology 3 domain of IRSp53/BAIAP2 in a GTP-Rho-dependent manner. The results suggest that IRSp53/BAIAP2 is a downstream effector of mDia1.

    Biochemical and biophysical research communications 2000;271;3;626-9

  • The insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of CNS synapses.

    Abbott MA, Wells DG and Fallon JR

    Department of Neuroscience, Brown University, Providence, Rhode Island 02912, USA.

    The synapse is the primary locus of cell-cell communication in the nervous system. It is now clear that the synapse incorporates diverse cell signaling modalities in addition to classical neurotransmission. Here we show that two components of the insulin pathway are localized at CNS synapses, where they are components of the postsynaptic density (PSD). An immunochemical screen revealed that polypeptides of 58 and 53 kDa (p58/53) were highly enriched in PSD fractions from rat cerebral cortex, hippocampus, and cerebellum. These polypeptides were purified and microsequenced, revealing that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Our analysis of IRSp58/53 mRNA suggests that within rat brain there is one coding region for IRSp58 and IRSp53; we find no evidence of alternative splicing. We demonstrate that IRSp58/53 is expressed in the synapse-rich molecular layer of the cerebellum and is highly concentrated at the synapses of cultured hippocampal neurons, where it co-localizes with the insulin receptor. Together, these data suggest that insulin signaling may play a role at CNS synapses.

    Funded by: NICHD NIH HHS: HD23924, R01 HD023924; NIMH NIH HHS: MH53571; NINDS NIH HHS: NS10343

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;17;7300-8

  • Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate.

    Okamura-Oho Y, Miyashita T, Ohmi K and Yamada M

    Department of Genetics, National Children's Memorial Medical Research Center, Taishido, Setagaya, Tokyo, Japan.

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis. Here, we describe a protein that binds to the DRPLA product. One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively. The longer form was mainly expressed in the brain and the shorter one in other tissues. The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1. Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs. A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding. An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays. The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1.

    Human molecular genetics 1999;8;6;947-57

  • Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1.

    Oda K, Shiratsuchi T, Nishimori H, Inazawa J, Yoshikawa H, Taketani Y, Nakamura Y and Tokino T

    Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

    BAI1 (brain-specific angiogenesis inhibitor 1) was originally isolated as a p53-target gene specifically expressed in brain. To clarify its function, we have been searching for cellular proteins that associate with the cytoplasmic domain of BAI1. Using its intracellular carboxyl terminus as "bait" in a yeast two-hybrid system, we isolated a cDNA clone named BAIAP2 whose nucleotide sequence would encode a 521-amino acid protein showing significant homology to a 58/53-kDa substrate of insulin-receptor kinase in the hamster. As the expression profile of BAIAP2 examined by Northern blot analysis was almost identical to that of BAI1, BAIAP2 appears to be active mainly in neurons. In vitro binding assays confirmed that a proline-rich cytoplasmic fragment of BAI1 interacted with the Src homology 3 (SH3) domain of BAIAP2. Double-color immunofluorescent analysis revealed that BAIAP2 was localized at the cytoplasmic membrane when it was coexpressed with BAI1 in COS-7 cells; BAIAP2 not associated with BAI1 was diffused in the cytoplasm. Predominant localization of BAI1 protein in a sub-cellular fraction enriched in growth cones indicated a possible role of BAI1 as a cell adhesion molecule inducing growth cone guidance. As a protein partner of BAI1, BAIAP2 may represent an important link between membrane and cytoskeleton in the process of neuronal growth.

    Cytogenetics and cell genetics 1999;84;1-2;75-82

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

Gene lists (7)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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