A Novel Actin Bundling/Filopodium-forming Domain Conserved in Insulin Receptor Tyrosine Kinase Substrate p53 and Missing in Metastasis Protein

Abstract

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.Insulin receptor tyrosine kinase substrate p53 (IRSp53), 1 also known as brain-specific angiogenesis inhibitor 1-associated protein 2, is a multifunctional adaptor protein enriched in the central nervous system (1-3). The protein contains a unique N-terminal 250-amino acid stretch, a half-Cdc42/Rac interactive binding (CRIB) motif, a proline-rich domain, a Src homology 3 (SH3) domain, and a WW domain-binding motif (WWB). IRSp53 is directly regulated by Rho family small GTPases Rac1 and Cdc42 and provides a molecular link between these GTPases and the actin cytoskeleton regulators WiskottAldrich syndrome protein (WASP) family verprolin homologous protein 2 (WAVE2) and mammalian enabled (Mena), which are involved in the formation of lamellipodia (4, 5) and filopodia (6, 7). Active Cdc42 binds to the half-CRIB motif (6, 7), whereas Rac1 binds to the unique N-terminal domain (8). The association of Rac1 or Cdc42 is proposed to liberate the C-terminal SH3 domain masked intramolecularly by its N terminus, thereby allowing the SH3 domain to interact with its binding partners (4, 7, 9). Thus, the SH3 domain is thought to be essential for IRSp53-mediated actin reorganization. However, the N-terminal half of IRSp53 lacking the SH3 domain was reported to induce neurite outgrowth in a neuroblastoma cell line (6) and filopodia in B16 melanoma cells (10), suggesting that IRSp53 promotes actin reorganization independently of SH3 domain-mediated intermolecular interactions.Recently, a novel monomeric actin-binding protein, missing in met...

The C Terminus of SipC Binds and Bundles F-actin to Promote Salmonella Invasion

“…Polymerization was initiated by adding actin polymerization buffer from a 50ϫ stock solution (100 mM MgCl 2 , 2.5 M KCl). F-actin cross-linking or bundling assays were carried out using the low-speed co-sedimentation assays as previously described (25). Briefly, monomeric rabbit G-actin was polymerized at room temperature in F-actin buffer (5 mM Tris-HCl at pH 7.8, 0.2 mM ATP, 1 mM dithiothreitol, 0.1 mM CaCl 2 , 1 mM MgCl 2 , and 100 mM KCl) for 1 h and preclarified at low-speed (10,000 ϫ g).…”

Filopodia: Complex models for simple rods

“…It will also be important to define how the IMD of IRSp53 family members contributes to filopodium formation, as multiple activities, like membrane binding, perhaps bending and tubulation or F-actin binding and bundling, have been attributed to this domain (Scita et al, 2008, Suetsugu et al, 2006. Moreover, IRSp53, but not the other family members, harbours a central CRIB (Cdc42/Rac interactive binding) motif rendering it a Cdc42 effector (Yamagishi et al, 2004). In spite of the wealth of information on structure and biological activities of the IMD as well as on potential binding partners of the SH3 domain, the particular molecular activities executed at a given sub-cellular position like lamellipodia or filopodia tips, or its exact interaction partners within the respective tip complexes remain obscure.…”

The Insulin Receptor Substrate of 53 kDa (IRSp53) Limits Hippocampal Synaptic Plasticity

“…GTPase binding to IRSp53 enables interactions of its SH3 domain with downstream effectors WAVE2, Mena, Eps8, or N-WASP, all of which are known regulators of actin dynamics (3)(4)(5)(6). In addition, the N-terminal IRSp53/missing in metastasis homology domain of IRSp53 assists in generating cellular protrusions by bundling actin filaments (5,7,8) and promoting membrane curvature (9,10). Expression of IRSp53 is particularly high in the brain, and consequently IRSp53 contributes to the formation of dendritic spines in the cultured hippocampal neuron model (11).…”