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FEBS Letters 586 (2012) 2790–2794

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Review Abi1, a critical molecule coordinating cytoskeleton reorganization with PI-3 kinase and growth signaling ⇑ Leszek Kotula

New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA

article info abstract

Article history: Coordination of actin cytoskeletal reorganization with growth and proliferation signals is a key cel- Received 4 April 2012 lular process that is not fully understood. PI-3 kinase is one of the central nodes for distributing Revised 10 May 2012 growth and proliferation signals downstream from growth factor receptors to the nucleus. Although Accepted 10 May 2012 PI-3 kinase function has been associated with actin cytoskeleton remodeling, satisfactory explana- Available online 19 May 2012 tions of the mechanisms mediating this regulation have been elusive. Here we propose that interac- Edited by Marius Sudol, Gianni Cesareni, tion of the Abi1 with the p85 regulatory subunit of PI-3 kinase represents the link between Giulio Superti-Furga and Wilhelm Just growth receptor signaling and actin cytoskeleton remodeling. This function of Abi1, which involves WAVE complex, was initially observed in macropinocytosis, and may explain the coincident dysreg- Keywords: ulation of PI-3 kinase and actin cytoskeleton in cancer. Actin cytoskeleton Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. WAVE complex Abi1 PI-3 kinase SH2 domain

1. Introduction 2. Regulation of WAVE complex and PI-3 kinase

Dysregulation of the actin cytoskeleton is associated with virtu- WAVE complexes are the major actin nucleation-promoting fac- ally all stages of human cancer regardless of origin [1]. The acqui- tors that regulate actin polymerization. In cells, WAVE complexes sition of specific knowledge of molecular mechanisms underlying consist of associations of ubiquitously expressed variants of 5 poly- human cancer is key to devising specific diagnostic and treatment peptides: Abl interactor-1 (Abi1, Hssh3bp1), Rac1-associated pro- strategies [2]. Delineation of precise mechanisms and of key tein 1 (Sra-1), Nck-associated protein 1 (Nap1), and WAVE2, as molecular players thus provides a rationale for future personalized well as a 9 kDa-peptide called Brck-1 or HSPC300 [7–9] (Fig 1B). treatment of individuals [3]. Among current advances, consider- Except for Brck-1, which is a single copy , each of these pro- able promise for anti-cancer treatment is offered by PI-3 kinase teins is member of a protein family consisting of two or three inhibitors [4,5]. Anti-proliferative effects of PI-3 kinase inhibitors in mammals including alternatively spliced isoforms of some genes have been associated with effects on actin cytoskeleton, suggesting [10]: Sra-1 and PIR121 (also known as Cyfip1 and Cyfip2), Nap1 that the cytoskeleton might be the important target for the thera- and Hem1, Abi1, Abi2, and Abi3 (also called NESH), and WAVE1, peutic effects of PI-3 kinase inhibitors in proposed therapies [5]. WAVE2, and WAVE3. Most of the isoforms have been shown to Although functional connections between actin cytoskeleton and assemble into WAVE-complexes in comparable fashion [11]. WAVE PI-3 kinase-dependent proliferation have been established, our complex has been implicated in membrane ruffling, but there is lack of knowledge of the mechanism makes it hard to explain or contradictory evidence as to which complexes regulate peripheral therapeutically exploit this phenomenon. Nonetheless, recent find- (lamellipodia) vs. dorsal ruffling [12,13]. WAVE complexes are also ings suggest that the regulation involves interaction of PI-3 kinase involved in cell motility and migration, cellular adhesion, cell- with WAVE complex (Fig. 1A), the major actin cytoskeleton regula- to-cell communication, cell division [9,14–17], and participate in tory complex, through its critical component, Abi1 [6] (Fig. 1B). immunological responses [18]. Thus, WAVE complexes are in- volved in numerous processes requiring major actin cytoskeleton reorganization and dynamics [19]. Importantly, dysregulation of WAVE complex or its components is associated with human cancer ⇑ Address: New York State Institute for Basic Research in Developmental [20–25]. Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314, USA. Fax: +1 718 698 0896. The critical role of Abi1 in dorsal ruffling identifies Abi1 as a E-mail address: [email protected] major regulatory protein in macropinocytosis. Macropinocytosis

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.05.015 L. Kotula / FEBS Letters 586 (2012) 2790–2794 2791

has been associated with invasiveness in prostate cancer [39,40], breast cancer [21], and epithelial tumorigenesis in general [22,23]. Recycling of integrins through PDGF-regulated dorsal ruf- fles [41], which likely occurs through direct binding of Abi1 to al- pha 4 integrin, as defined in cells from the conventional Abi1 KO mouse [42], links regulation of macropinocytosis to directional cel- lular migration and cellular adhesion, and thus, to processes that have been significantly implicated in cancer tumorigenesis and progression. PI-3 kinase is dysregulated in human cancer, and its dysregula- tion contributes to tumor progression. Thus, it is important to understand the mechanism of its regulation [43,44]. PI-3 kinases can be divided into two subclasses, class IA PI-3 kinases and class IB PI-3 kinases. Class IA PI-3 kinases are activated by receptor tyro- sine kinases, whereas class IB PI-3 kinases are activated by G-pro- tein-coupled receptors [45]. Here, we are concerned with class IA PI-3 kinases. Recent studies have shed considerable light on the regulation of class IA PI-3 kinase activity [46]. PI-3 kinase activity is regulated by autoinhibitory interactions between its subunits, p110 and p85 [47]. The catalytic activity is mediated by p110, p85 is the regulatory subunit. Structural studies have confirmed earlier functional observations that identified inhibitory interac- tions between catalytic p110 and regulatory p85 subunits [48]. These interactions involve sub-domains of p85 including iSH2, C- SH2, and N-SH2 domains, and sub-domains of p110 including C2, helical, and the catalytic (kinase) domains (Fig. 1C). Interactions between the C-SH2 domain of p85 and the catalytic subunit dif- fered depending on the isoform of the kinase [49,50]. In particular, the interaction of the p85 C-SH2 domain with the p110 kinase do- Fig. 1. Structural determinants of WAVE and PI-3 kinase complexes. (A) Anatomy of WAVE complex. WAVE complex integral include Rac1-associated protein 1 main occurs in PI-3 kinase beta, but not in PI-3 kinase alpha. How- (Sra-1), Nck-associated protein 1 (Nap1), Abl interactor-1 (Abi1, Hssh3bp1), and ever, the interaction between the p85 iSH2 domain and the p110 WAVE2, and Brck-1 or HSPC300 or their variants. Activation of WAVE complex C2 domain is weaker in PI-3 kinase beta than in PI-3 kinase alpha occurs upon conformational change leading to availability of VCA to bind Arp2/3 [51]. Because of this latter observation, and despite the presence of complex. WAVE complex has been implicated in multiple actin polymerization- dependent cellular functions. (B) Domain structure of Abi1. Abi1 structural and the additional inhibitory C-SH2-kinase domain interaction, there is functional domains and identified binding regions for listed proteins or their controversy as to whether PI-3 kinase beta is more constitutively domains. WAVE-BD, WAVE domain binding region [64,77]; HHR, homeo-domain active than PI-3 kinase alpha [47]. Moreover, for the beta isoform, homologous region; Abl-SH3-SH2, c-Abl kinase SH3-SH2 domain binding region ligands that disrupt the interaction between the C-SH2 domain and [65], PEST, PXXP-, Ser–rich, or PolyPro-rich, sequences rich in SH3 domain binding the catalytic domain have the potential to activate PI-3 kinase, as consensus PXXP, serine or proline residues, respectively; SH3, Src homology 3 domain. Indicated binding sites for: Spectrin-SH3, Spectrin Src Homology 3 domain; has been proposed for the N-SH2 domain in both PI3 kinase iso- Eps8-SH3, Eps8 Src Homology 3 domain; p85-N-SH2, p85 regulatory subunit of forms [52]. Data from our laboratory [6] and others [66] suggest phosphoinositide-3 kinase; Abl-PRL, c-Abl kinase proline rich region, N-WASP and that members of the Abi protein family fit the paradigm for such mDia. Black arrows indicate identified tyrosine phosphorylation sites: pY213, regulatory ligands. We postulate that Abi1 regulates PI-3 kinase pY421, pY435, and pY506, which were identified to interact with p85-SH2 domains [6,66]. (C) Autoinhibitory structure of PI-3 kinase. Linear depiction of inhibitory activity through regulating the p85 SH2 domain interaction with interaction between subunits of PI-3 kinase. Top, p110 catalytic subunit (alpha or the catalytic p110 subunit. beta) of PI-3 kinase and its subdomains: ABD, adaptor binding domain; RAS, Ras WAVE complex integrity is central to its regulatory role in pro- binding domain; C2, C2 domain; Helical, helical domain; and Kinase, kinase/ moting actin polymerization. Several candidate mechanisms of catalytic domain. Bottom, p85 regulatory subnit of PI-3 kinase and its subdomains: regulation of WAVE complex have been proposed, all of which in- SH3, Src homology 3 domain; RhoGAP, Rho GTPase-related domain; N-SH2, N- terminal Src homology 2 domain; iSH2, inter-SH2 domain and C-SH2, C-terminal volve integrity of the complex requiring the presence of all constit- Src homology 2 domain. Arrows indicate inhibitory interactions between the uents. This conclusion was confirmed by in vitro reconstitution subunit subdomains. p110 beta has additional interaction involving the C-SH2 and studies of WAVE complex [53,54], siRNA studies in cultured cells kinase domain but weaker iSH2-C2 interaction. Abi1 phosphopeptides can bind N- [55,56], and in cells with genetic inactivation of WAVE [57] or SH2 and C-SH2 in vitro [6] raising the possibility of PI-3 kinase activation by competing off- and thus alleviating with the inhibitory interaction imposed by p85 Abi1 genes [38]. Downregulation or removal of any integral WAVE subunit. complex protein leads to lower levels of specific WAVE complexes, which results in reduced WAVE activity as assessed by directly measuring actin polymerization in vitro, or by cellular motility is an actin cytoskeleton-dependent process characterized by exten- and peripheral and dorsal ruffle formation assays. sive plasma membrane reorganization involving membrane ruf- Actin polymerization requires active WAVE complex. Activation fling followed by formation of an extended and polymerized of WAVE complex involves a conformational change of inactive actin-rich structure, which is subsequently enclosed and internal- WAVE complex that renders the VCA domain available for interac- ized as a macropinosome. Macropinocytosis is involved in nutrient tion with Arp 2/3 [58,59]. Structural studies have revealed that the uptake [26]; It is normally upregulated following growth factor conformational change that releases intermolecular VCA from and mitogenic agent stimulation [27–29] and pathologically sequestration is induced by Rac-GTP binding to an allosteric site upregulated in cancer cell lines [30–37]. Fibroblasts lacking Abi1 on the WAVE complex [58]. Full activation of WAVE complex re- have a significant defect in dorsal ruffling and have downregulated quires prenylated, activated Rac1 and the presence of acidic phos- WAVE2 and WAVE1 complexes, but have upregulated Abi2 and pholipids such as PIP3 [54]. PIP3 binds directly to the basic region WAVE3 [38]. Dysregulation of WAVE complexes or its components of WAVE [60,61]. In addition to promoting activity of WAVE com- 2792 L. Kotula / FEBS Letters 586 (2012) 2790–2794

Fig. 2. Regulation of WAVE and PI-3 kinase by Abi1. (A) A model for regulation of WAVE complex by phosphotyrosine-dependent interaction of Abi1 with p85. Active WAVE complex promotes actin polymerization upstream of Arp2/3 complex. Under non-phosphorylated conditions, WAVE complex is inactive, and all components are bound within the complex. Activation of the complex requires binding of active, prenylated Rac1 and acidic phospholipids (such as PIP3), as well as serine phosphorylation of Abi1 [54]. The latter occurs downstream of Erk, which promotes WAVE complex activity [63]. Abl kinase is proposed to have a negative effect on WAVE complex activity in vitro [64], although WAVE2 when phosphorylated on tyrosine 150 by Abl rescues membrane ruffling [78]. Abl-mediated phosphorylation of Abi1 leads to recruitment of Abi1 by p85, which occurs through binding of phospho-Abi1 to N-SH2 and C-SH2 domains [6]. We demonstrated the feasibility of the WAVE to p85 shuttling hypothesis by expressing an Abi1 Y213F mutant, which led to enhanced macropinocytic uptake and cell spreading activity in comparison to wild type controls [6]. We postulate that this result is due to an increase in active WAVE complex as Abi1 Y213F, because of its reduced affinity for p85, is shifted into WAVE2 complex. (B) Diagram depicting a hypothetical mechanism of reciprocal regulation of WAVE2 complex-dependent and PI-3 kinase pathways. The presence of Abi1 within intact WAVE complex promotes its function in actin polymerization-dependent processes such as macropinocytosis and cellular adhesion. c-Abl-dependent tyrosine phosphorylation [6,65] leads to recruitment of phospho-Abi1 into a complex with p85 through an interaction with the p85 SH2 domain [6] leading to increased PI-3 kinase activity. This leads to enhanced levels of phospho-Akt resulting in enhanced cellular proliferation. plex, PIP3 is thought to help maintain membrane localization of 213 (pY213) also regulates Abl kinase activity [65]. Abi1-pY213 the activated WAVE complex [60]. It has been proposed that Arf transmits downstream signals through interaction with PI-3 kinase cooperates with Rac1 to activate WAVE complex, but the mecha- regulatory subunit p85 [6]. nism of this regulation has not been fully elucidated [62]. Analysis of an Abi1-Y213F mutant revealed a mechanism by Phosphorylation regulates WAVE complex and Abi1 function. which Abi1 might regulate macropinocytosis. The Y213F mutation Serine phosphorylation of WAVE and Abi proteins has been found blocked the interaction of Abi1 with the p85 subunit of PI-3 kinase, in active WAVE complex suggesting a possible role of serine phos- resulting in enhanced macropinocytic uptake and abrogation of the phorylation in WAVE activation [54]. This observation is consistent ability of Abl kinase to regulate uptake [6]. Of interest, tyrosine with the finding that Erk-dependent serine phosphorylation of phosphorylated Abi1 has not been detected in active WAVE com- Abi1 promotes cell motility [63]. The role of tyrosine phosphoryla- plex [54]. Thus, we propose that non-tyrosine-phosphorylated tion in WAVE complex activation is not well defined. Tyrosine- Abi1 associates with WAVE complex, but tyrosine-phosphorylated phosphorylated Abi1 was not observed in the studies by Lebensohn Abi1 does not (Fig. 2A). Upon tyrosine phosphorylation, Abi1 asso- reporting serine phosphorylation. The action of c-Abl tyrosine ki- ciates with the p85 subunit of PI-3 kinase through several phos- nase was reported to abrogate WAVE complex in vitro [64], but photyrosine interactions, which we identified by unbiased SH2 the mechanism of this regulation has not been determined. In con- domain screening [6]. We postulate that the Abi1-p85 interaction trast, phosphorylation of WAVE2 at tyrosine 150 was proposed to competes with the inhibitory interaction of p85 with the catalytic promote activation of WAVE complex through an allosteric effect subunit of PI-3 kinase, as recently confirmed for phosphopeptides relieving VCA inhibition [58]. The role of Abi1 tyrosine phosphory- interacting with the p85-SH2 domain [50]. This hypothesis to ex- lation in WAVE complex regulation is possibly even more complex. plain regulation of PI-3 kinase by Abi1 is consistent with results Abi1 is phosphorylated on several tyrosine residues in an Abl ki- from Abi1 siRNA knockdown studies, which demonstrated that nase-dependent manner [65,66]. Phosphorylation of Abi1 tyrosine downregulation of Abi1 (putatively resulting in decreased PI-3 ki- L. Kotula / FEBS Letters 586 (2012) 2790–2794 2793 nase activity) causes decreased levels of phosphorylated Akt in to macropinocytosis, cell-to-cell adhesion requires WAVE function breast cancer cell lines [67]. Consistently with the proposed regu- [75,76]. PI-3 kinase activity is required for coincidental prolifera- lation, enhanced levels of phospho-Akt also correlate with high tion and mitogenic signaling through PIP3 production. Under- Abi1 expression in invasive breast cancer [21]. standing the role of Abi1 in these regulatory processes should help to define mechanisms for coordinated actin polymerization 3. The shuttling hypothesis and cellular proliferation, which are required for proper spatiotem- poral regulation of mitogenic and actin-dependent signaling Tyrosine phosphorylation of Abi1 potentially regulates WAVE events, which are dysregulated in human cancer. and PI-3 kinase complexes. The emerging consensus is that Abi1 participates in complexes with WAVE and PI-3 kinase p85. Based Acknowledgments on our results [6] and taking into consideration protein–protein interactions, we postulate that interaction of p85 with Abi1 plays I would like to thank Kazuya Machida, Bruce Mayer, Joel an inhibitory role in actin polymerization. The interaction of p85 Swanson and Jonathan Backer for helpful discussions. I am grateful and Abi1, which is Abl kinase-dependent and phosphotyrosine- to Ted Brown and Richard Carp (IBR, Staten Island) for their support dependent, occurs through the binding of the p85 SH2 domain to and encouragement. Supported by grants from Department of De- tyrosine phosphorylated Abi1. Our single-site affinity analysis fense Prostate Cancer Research Program (W81XWH-08-1-0320) suggests that the interaction between Abi1 and p85 involves coop- (LK) and in part by NIH R01 NS044968 (LK). Support by the FM Kir- erative binding of multiple Abi1 phosphotyrosines with sites in the by Foundation, Inc., Morristown, NJ (LK) is acknowledged. 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