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Characterisation of IRTKS, a Novel Irsp53/MIM Family Actin Regulator with Distinct Filament Bundling Properties

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Characterisation of IRTKS, a Novel Irsp53/MIM Family Actin Regulator with Distinct Filament Bundling Properties Research Article 1663 Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties Thomas H. Millard*, John Dawson and Laura M. Machesky‡ School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK *Present address: University of Bristol, Dept. of Biochemistry, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK ‡Author for correspondence (e-mail: [email protected]) Accepted 12 March 2007 Journal of Cell Science 120, 1663-1672 Published by The Company of Biologists 2007 doi:10.1242/jcs.001776 Summary IRSp53 is a scaffold protein that contains an IRSp53/MIM resembles a WASP-homology 2 (WH2) motif. Addition of homology domain (IMD) that bundles actin filaments and the Ct extension to IRSp53 causes an apparent shortening interacts with the small GTPase Rac. IRSp53 also binds to of bundles induced by the IMD in vitro, and in cultured the small GTPase Cdc42 and to Scar/WAVE and cells, suggesting that the Ct extension of IRTKS modulates Mena/VASP proteins to regulate the actin cytoskeleton. We the organising activity of the IMD. Lastly, we could not have characterised a novel IMD-containing protein, insulin detect actin monomer sequestration by the Ct extension of receptor tyrosine kinase substrate (IRTKS), which has IRTKS as would be expected with a conventional WH2 widespread tissue distribution, is a substrate for the insulin motif, but it did interact with actin filaments. receptor and binds Rac. Unlike IRSp53, IRTKS does not interact with Cdc42. Expression of IRTKS induces clusters Supplementary material available online at of short actin bundles rather than filopodia-like http://jcs.biologists.org/cgi/content/full/120/9/1663/DC1 protrusions. This difference may be attributable to a short carboxyl-terminal (Ct) extension present on IRTKS, which Key words: GTPase, Actin, Cytoskeleton, Filopodia, Motility Introduction of the signals that control the formation of filopodia. Journal of Cell Science The actin cytoskeleton is central to the movement, morphology Activation or overexpression of the GTPase Cdc42 induces and adhesion of eukaryotic cells (Pollard and Borisy, 2003). filopodia in some cell types, but the key downstream effectors Actin filaments are used in a diverse array of cellular contexts of Cdc42-induced filopodia are not known (Lamarche et al., and numerous actin binding proteins and regulators control the 1996; Nobes and Hall, 1995). There is increasing evidence that polymerisation and arrangement of filaments. In motile cells, Ena/VASP proteins are involved in filopodia formation, acting polymerisation of actin at the leading edge drives protrusion of by preventing filament capping, resulting in the production of the membrane. Protrusive actin structures include lamellipodia, long parallel filaments (Bear et al., 2002; Lebrand et al., 2004; which are broad, flat sheet-like projections, and filopodia, Mejillano et al., 2004). Diaphanous related formins (DRF) which are thin, needle-like projections (Small et al., 2002). proteins are also key controllers of filopodia formation Lamellipodia consist of a dense network of short, branched (Pellegrin and Mellor, 2005; Schirenbeck et al., 2005a; actin filaments, and our understanding of how these structures Schirenbeck et al., 2006; Schirenbeck et al., 2005b). In are formed have seen a dramatic increase in recent years addition, numerous actin bundling and crosslinking proteins (Svitkina and Borisy, 1999). Central to lamellipodia formation control the arrangement of actin filament protrusions (Revenu is the Arp2/3 complex, which nucleates actin filaments while et al., 2004; Vignjevic et al., 2006). Bundling is a crucial bound to the side of existing filaments, resulting in a branched determinant of the mechanical properties of actin protrusions filament network (Mullins et al., 1998). Signals are transduced (Gardel et al., 2004; Xu et al., 1998). Different actin structures from extracellular stimuli to the Arp2/3 complex by a pathway contain different complements of bundling proteins, for including the small GTPase Rac and Scar (WAVE) proteins instance fascin is found within filopodia and filamin A within (Bompard and Caron, 2004; Machesky and Insall, 1998). lamellipodia (Flanagan et al., 2001; Kureishy et al., 2002). Filaments in lamellipodia are short due to the presence of Like the proteins that control actin assembly, bundling proteins capping protein, which binds to the barbed end of growing are subject to regulation and it is becoming apparent that in filaments shortly after nucleation, preventing further growth order to generate specific actin structures, actin assembly and (Pollard and Borisy, 2003). In contrast to lamellipodia, bundling must be co-ordinately controlled (Revenu et al., filopodia consist of a combination of long, unbranched 2004; Vignjevic et al., 2006). filaments arranged in tight, parallel bundles (Svitkina et al., Insulin receptor substrate of 53 kDa (IRSp53) was originally 2003) and shorter unbundled filaments near the tips (Medalia identified as a phosphorylation substrate for the insulin et al., 2007). We currently do not have a clear understanding receptor, however, subsequently research has focused on its 1664 Journal of Cell Science 120 (9) role in regulating the actin cytoskeleton (Yeh et al., 1996). isoform 2 or IRSp53-S, NM_017450). This sequence has Several groups have observed that overexpression of IRSp53 previously been identified by Yamagishi et al. who referred to results in the formation of filopodia-like protrusions, and it as IRTKS in sequence alignments (Yamagishi et al., 2004). IRSp53 localises to the tips of both filopodia and lamellipodia Homologous sequences have also been identified for several (Govind et al., 2001; Krugmann et al., 2001; Nakagawa et al., other species, including mouse (NP_080109, 87% identity), 2003; Yamagishi et al., 2004). IRSp53 is a modular protein chicken (XP_414749, 70% identity) and zebrafish containing several protein interaction domains, including an (AAH68330, 52% identity). The human IRTKS gene is located SH3 domain, which binds to several important actin regulators on chromosome 7 at q21.3–q22.1, whereas IRSp53 is on including Scar2/WAVE2 and the Ena/VASP protein, Mena chromosome 17 at q25. (Krugmann et al., 2001; Miki et al., 2000). IRSp53 also Alignment of human IRTKS with IRSp53 demonstrates that possesses distinct binding sites for the two Rho family the region corresponding to the IMD of IRSp53 is well GTPases, Rac and Cdc42, leading to the proposal that IRSp53 conserved, as are the SH3 domain and WW domain interaction functions as an adaptor, transducing signals from these motif (Fig. 1). Outside of these regions conservation is low, GTPases to cytoskeletal regulating proteins (Krugmann et al., with IRTKS notably lacking the partial CRIB domain which 2001; Miki et al., 2000). However, recent work has suggested mediates binding of Cdc42 to IRSp53. The C terminus of that IRSp53 also possesses effector functions (Yamagishi et al., IRTKS has no similarity to that of the well-characterised short 2004). The N-terminal 250 amino acids of IRSp53 consist of (S) splice variant of IRSp53, however, it does have homology a domain conserved in five mammalian proteins including the to the C-termini of two other IRSp53 splice variants, the L previously identified actin regulator missing in metastasis-B (long) and M (medium) forms (Fig. 1B) (Miyahara et al., 2003; (MIM-B), hence the name IRSp53/MIM homology domain Yamagishi et al., 2004). Note that unless otherwise stated, (IMD). The crystal structure of the IRSp53 IMD reveals it to IRSp53 refers to the S splice variant in this manuscript. be a zeppelin-shaped dimer with one F-actin-binding site per monomer (Millard et al., 2005). In isolation the IMD can IRTKS is widely distributed and is an insulin receptor bundle actin filaments and induce filopodia-like protrusions, substrate however, mutation of the actin binding sites abrogates We used peptides based on the sequence of human and mouse protrusion formation by both the isolated IMD and by full- IRTKS (see Materials and Methods) to raise a polyclonal length IRSp53 (Millard et al., 2005). The structure of the IMD antibody, which recognised a band of 60 kDa in extracts from is closely related to that of the BAR domain, a function of the mouse myoblast cell line C2C12 (see Fig. S1A in which is to tubulate membranes (Gallop and McMahon, 2005). supplementary material). Immunoprecipitations from C2C12 Intriguingly, recent data has indicated that the IRSp53 IMD extract were performed using this antibody and the precipitated also has the capacity to tubulate membranes and it has been material subjected to SDS-PAGE. A band of 60 kDa was suggested that this activity contributes to protrusion formation excised from the gel and analysed by mass spectrometry after (Suetsugu et al., 2006). Like IRSp53, MIM-B can bundle trypsin digestion. Three of the five peptide sequences derived filaments via its IMD, however, outside the IMD, MIM-B has were found to match the murine form of the novel protein (Fig. Journal of Cell Science little sequence conservation with IRSp53, suggesting a S1B in supplementary material). The tissue distribution of functional divergence (Yamagishi et al., 2004). Unlike IRSp53, IRTKS was studied by immunoblotting murine tissue extracts. MIM-B does not possess an SH3 domain, instead it contains Western blots were inconclusive as the IRTKS antibody an actin monomer binding WASP homology 2 (WH2) domain produced
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