Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2–Abi1–Nap1–PIR121 complex. The WAVE2–Abi1–Nap1–PIR121 complex is as active as the WAVE2–Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

[1]  T. Pollard.  The cytoskeleton, cellular motility and the reductionist agenda , 2003, Nature.

[2]  A. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[3]  J. Condeelis,et al.  Cofilin Produces Newly Polymerized Actin Filaments that Are Preferred for Dendritic Nucleation by the Arp2/3 Complex , 2002, Current Biology.

[4]  Josephine C. Adams,et al.  Regulation of protrusive and contractile cell-matrix contacts. , 2002, Journal of cell science.

[5]  T. Pawson,et al.  The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling , 2002, Nature Cell Biology.

[6]  Alexandre V. Podtelejnikov,et al.  Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck , 2002, Nature.

[7]  M. Kasuga,et al.  Molecular cloning of p125Nap1, a protein that associates with an SH3 domain of Nck. , 1996, Biochemical and biophysical research communications.

[8]  N. Nomura,et al.  p140Sra-1 (Specifically Rac1-associated Protein) Is a Novel Specific Target for Rac1 Small GTPase* , 1998, The Journal of Biological Chemistry.

[9]  J. Mandel,et al.  A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[10]  C. Betsholtz,et al.  EPS8 and E3B1 transduce signals from Ras to Rac , 1999, Nature.

[11]  R. Iggo,et al.  Increased apoptosis induction by 121F mutant p53 , 1999, EMBO Journal.

[12]  M. Suyama,et al.  Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. , 1998, DNA research : an international journal for rapid publication of reports on genes and genomes.

[13]  P. G. Allen.  Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles , 2003, Nature Cell Biology.

[14]  S. Goff,et al.  Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity. , 1995, Genes & development.

[15]  Zhenbiao Yang,et al.  RHO Gtpases and the Actin Cytoskeleton , 2000 .

[16]  C. Mello,et al.  The GEX-2 and GEX-3 proteins are required for tissue morphogenesis and cell migrations in C. elegans. , 2002, Genes & development.

[17]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[18]  P. Gounon,et al.  The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins , 2000, Nature Cell Biology.

[19]  R. Vale,et al.  Molecular requirements for actin-based lamella formation in Drosophila S2 cells , 2003, The Journal of cell biology.

[20]  A. Pendergast,et al.  Abi-2, a novel SH3-containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity. , 1995, Genes & development.

[21]  Y. Sakaki,et al.  Molecular cloning of a novel apoptosis-related gene, human Nap1 (NCKAP1), and its possible relation to Alzheimer disease. , 2000, Genomics.

[22]  Y. Sakaki,et al.  Isolation of hNap1BP which interacts with human Nap1 (NCKAP1) whose expression is down-regulated in Alzheimer's disease. , 2001, Gene.

[23]  M. Mann,et al.  In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly , 1998, The EMBO journal.

[24]  M. Kasuga,et al.  Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac. , 1997, The Biochemical journal.

[25]  T. Takenawa,et al.  WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement. , 2001, Journal of cell science.

[26]  C. Piccoli,et al.  Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth , 1997, Oncogene.

[27]  T. Takenawa,et al.  IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling , 2000, Nature.

[28]  K. Rottner,et al.  Sra‐1 and Nap1 link Rac to actin assembly driving lamellipodia formation , 2004, The EMBO journal.

[29]  J. Vacher,et al.  Characterization and expression analyses of the mouse Wiskott-Aldrich syndrome protein (WASP) family member Wave1/Scar. , 2002, Gene.

[30]  J. Raber,et al.  Loss of WAVE-1 causes sensorimotor retardation and reduced learning and memory in mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.