Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation

Abstract

WAVE2 is a member of the Wiskott-Aldrich syndrome protein family of cytoskeletal regulatory proteins shown to link Rac activation to actin remodeling via induction of Arp 2͞3 activity. WAVE2 is thought to be regulated by its positioning in a macromolecular complex also containing the Abelson-(Abl) interactor-1 (Abi-1) adaptor, but the molecular basis and biologic relevance of WAVE2 inclusion in this complex are ill defined. Here we show that Abi-1 binding to WAVE2 is mediated by discrete motifs in the Abi-1 coiled-coil and WAVE2 WAVE-homology domains and increases markedly in conjunction with Abi-1-WAVE2 translocation and colocalization at the leading edge in B16F1 cells after fibronectin stimulation. Abi-1 also couples WAVE2 to Abl after cell stimulation, an interaction that triggers Abl membrane translocation with WAVE2, Abi-1, and activated Rac, as well as Abl-mediated tyrosine phosphorylation and WAVE2 activation. By contrast, mutation of tyrosine residue Y150, identified here as the major site of Ablmediated WAVE2 tyrosine phosphorylation, as well as disruption of WAVE2-Abi-1 binding, impairs induction of WAVE2-driven actin polymerization and its membrane translocation in association with activated Rac. Similarly, WAVE2 tyrosine phosphorylation and induction of membrane actin rearrangement are abrogated in fibroblasts lacking the Abl family kinase. Together, these data reveal that Abi-1-mediated coupling of Abl to WAVE2 promotes Abl-evoked WAVE2 tyrosine phosphorylation required to link WAVE2 with activated Rac and with actin polymerization and remodeling at the cell periphery.actin polymerization ͉ cytoskeletal dynamics ͉ WASp family T he actin cytoskeleton is a dynamic structure integrally involved in the coupling of extracellular stimuli to cell activation and concomitant changes in morphology, motility, and many other fundamental cellular processes. Among the many effectors involved in poststimulatory actin-based cellular responses, members of the Wiskott-Aldrich syndrome protein (WASp) family are now recognized as particularly important players in connecting stimulatory signals to actin cytoskeletal reorganization. In addition to WASp, this family includes the WASp ortholog, N-WASp, and, in mammalian cells, three WAVE͞SCAR isoforms, WAVEs 1, 2, and 3 (1-3). All of these cytosolic proteins contain a C-terminal verprolin homology͞connecting region͞acidic region (VCA) domain, which mediates binding to actin monomers and the Arp 2͞3 complex and consequent induction of Arp 2͞3 actin nucleating activity (4). Immediately upstream of the VCA domain, the WASp family members all contain a proline-rich region that mediates interaction with profilin and SH3 domain-containing proteins, the latter of which modulate subcellular location and possibly activation of WASp͞N-WASp (5-7). WASp and N-WASp also contain an N-terminal EVH1 and cdc42͞Rac interactive binding domain, which respectively mediate interactions with the WASp-interacting protein and the activated form of the Rho family GTPase, cdc42 (8, 9). WASp͞N-WASp ...

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

“…For example, in leukocytes, Abi1 was found in the WAVE2 complex and Abi2 in WAVE1 complex, whereas both Abi1 and Abi2 were present in WAVE2 complex in A431 cells (20). The basis for specificity of Abi protein incorporation into WAVE1 or WAVE2 complex is not known, although, based on high sequence conservation of binding sites, the affinities of the Abi proteins for WAVE proteins are expected to be similar (20,22,31). One explanation might be that Abi1 and Abi2 exhibit different patterns of threonine and serine phosphorylation (20), which is required for full activation of WAVE2 complex by Rac and acidic phospholipids (20).…”

“…Rac1 does not interact directly with WAVE but instead binds to the Nap1-binding protein Sra-1 (12) and to Abi1 (21). The Abi1-mediated tyrosine phosphorylation of WAVE2 by Abl regulates WAVE2 complex activity either by regulating conformation of the complex, as suggested (22), or by regulating interactions among the complex components or with other cellular targets (10,23,24). The interaction of Abi1 with p85 (24) might modulate PI3 kinase activity (10); this activity would be consistent with coincident modulation of WAVE complex activity by its interaction with PIP3, or PIP3-mediated membrane recruitment of WAVE2 (25).…”

c-Abl-mediated Phosphorylation of WAVE3 Is Required for Lamellipodia Formation and Cell Migration

“…Westphal and colleagues (32) showed that c-Abl interacts with the WAVE protein complex. Furthermore, c-Abl tyrosine kinase activity was found to be required for the phosphorylation of WAVE2, a close relative of WAVE3 (30). Next, we investigated whether WAVE3 and Abl interact endogenously in MDA-MB-231 cells.…”

“…On the other hand, it has been shown that Ablmediated phosphorylation of WAVE2 is mediated by Abi1 in an Abl⅐Abi1⅐WAVE2 complex (30). To test whether Abi1 is required for coupling of Abl to WAVE3 to promote Abl-mediated phosphorylation of WAVE3, we analyzed WAVE3 phosphorylation by Abl both in the presence and absence of Abi1.…”

“…c-Abl has been found to regulate cell motility and neurite extension and to localize to specific actin structures, such as focal adhesions, and lamellipodia (27)(28)(29). Ablmediated tyrosine phosphorylation of WAVE2 was found to greatly enhance cell migration and adhesion, and both Abl and WAVE2 localize to the cell periphery upon growth factor stimulation (30).…”