The N-Terminal Half of Abl Family Kinases Is Highly Conserved

The Abl kinase family is composed of the Abl and Arg (Abl-related gene, aka Abl2) kinases in vertebrates,^1,2 the Abl kinase in flies,³ and the Abl-1 kinase in worms.⁴ Following a short variable region, Abl family kinases contain tandem Src homology (SH) 3, SH2, and tyrosine kinase domains (Fig. 1). The SH3 and SH2 domains regulate kinase activity. In the inactive state, the SH3 and SH2 domains form an inhibitory scaffold along the back face of the kinase domain that holds it in the “off ” state.^5,6 Upon activation, the molecule undergoes a conformational change in which the SH3 and SH2 are relieved from their inhibitory conformation and promote kinase activity by facilitating interactions with substrates.^7,8 The Chapter by Hantschel and Superti-Furga contains an excellent review of Abl kinase regulation.

Figure 1

Domain stuctures of Abl, Arg, fly Abl, and Bcr-Abl. At least two alternatively-spliced amino termini have been reported for Abl and Arg. Only the myristoylated (myr) form (type IV for Abl, type 1B for Arg) is shown here. Abl (mouse): Following the N-terminal (more...)

The C-Terminal Halves of Abl Family Kinases Contain Actin and Microtubule-Binding Domains

Abl family kinases share less sequence conservation downstream of the tyrosine kinase domain. The functional features of the C-terminal half are most well-characterized in Abl and Arg. Immediately C-terminal to the kinase domain, Abl and Arg contain three conserved Pro-X-X-Pro (PXXP) motifs that serve as binding sites for SH3-domain-containing proteins^9-13 (Fig. 1). The C-terminal halves of Abl family kinases are unique among nonreceptor tyrosine kinases because they contain multiple domains that interact directly with actin and microtubules. Both Abl and Arg share C-terminal calponin homology F-actin-binding domains^14-16 (Fig. 1). A globular (G-) actin-binding domain precedes this F-actin-binding domain in Abl,¹⁵ whereas in Arg, it is preceded by an I/LWEQ homology F-actin-binding domain and a microtubule-binding domain.^16,17 Based on sequence comparisons, the fly Abl and Abl-1 proteins in worms are likely to contain an F-actin binding domain at their C-termini, but the actin-binding properties of these family members have not yet been examined.

Abl Contains Nuclear Localization and Export Sequences

In addition to the features that Abl shares with Arg, the C-terminal half of Abl also contains a DNA-binding domain with 3 HMG-like motifs,¹⁸ 3 nuclear localization sequences,¹⁹ and a nuclear export sequence²⁰ (Fig. 1). These features appear to be important for Abl to function in the cell nucleus. The Chapter by Wang, Minami, and Zhu contains an excellent review of Abl's nuclear functions.

The Bcr-Abl Oncoprotein

Mutant forms of Abl cause chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) in humans. Oncogenic activation of Abl is most commonly associated with a chromosomal translocation that fuses sequences encoding a portion of the Bcr protein to an N-terminal part of Abl resulting in a hybrid gene encoding the Bcr-Abl oncoprotein.²¹ A coiled-coil motif in Bcr promotes oligomerization of the Bcr-Abl fusion protein^22,23 (Fig. 1). This oligomerization promotes kinase autophosphorylation and activates Abl tyrosine kinase activity,^24-26 leading to the activation of anti-apoptotic and mitogenic signaling pathways.^27-29 Oligomerization also promotes binding of Bcr-Abl to F-actin stress fibers,²⁴ which may contribute to the alterations in cytoskeletal structure³⁰ and cell adhesion systems in Bcr-Abl-transformed cells.³¹

Abl and Arg Promote Cell Protrusions in Response to Growth Factors or Adhesive Cues

The first evidence that Abl family kinases regulate actin-based protrusions of the cell membrane came from observations of Bcr-Abl-transformed cells.³⁰ Salgia and colleagues showed that Bcr-Abl-transformed NIH3T3 fibroblasts exhibit more filopodia, lamellipodia, and membrane ruffles than their untransformed counterparts.³⁰ In contrast to the normal smooth round appearance of BaF3 hematopoietic cells, Bcr-Abl-expressing BaF3 cells extended numerous pseudopodia and were hypermotile on various adhesive surfaces.³⁰ Despite this clear demonstration that an activated form of Abl could promote changes in cytoskeletal structure, it was unclear how this activity related to the normal functions of Abl or Arg.

Subsequent studies of Abl- and/or Arg-deficient fibroblasts have clearly demonstrated that Abl and Arg act downstream of growth factor and adhesion receptors to promote cytoskeletal rearrangements. Application of platelet-derived growth factor (PDGF) to fibroblasts leads to dramatic actin-based ruffling of the cell periphery.^32,33 The finding that PDGF induces Abl kinase activity led Plattner and Pendergast and colleagues to examine a role for Abl in membrane ruffling (Fig. 2A). Abl-deficient fibroblasts exhibit dramatically reduced membrane ruffling in response to PDGF, but this response can be restored by Abl reexpression.³³ These studies were the first to demonstrate that a normal function of Abl is to coordinate morphogenetic responses to a specific extracellular cue. Subsequent studies have further confirmed that Abl family kinases are required for membrane ruffling following cell exposure to PDGF and epithelial growth factor (EGF).³⁴ Although the function of these growth factor-induced ruffles is unknown, they may promote cell migration by helping cells sample three-dimensional space.

Figure 2

Abl family kinases are required for cellular morphogenesis. A) Top: treatment of wild type fibroblasts with PDGF induces multiple F-actin-rich ruffles. Middle: abl^-/- fibroblasts treated with PDGF fail to exhibit ruffling. Bottom: reexpression of Abl (more...)

Abl family kinases also mediate cytoskeletal rearrangements in response to integrin-mediated adhesion to extracellular matrix proteins. Wild type fibroblasts are highly dynamic as they attach and spread on fibronectin-coated surfaces, frequently extending and retracting actin-based lamellipodial and filopodial protrusions.¹⁷ Kymographic analysis reveals that Arg-deficient fibroblasts exhibit significantly fewer lamellipodial protrusions and retractions than wild type fibroblasts during adhesion and spreading on fibronectin (Fig. 2B). Reexpression of a functional Arg-yellow fluorescent protein (Arg-YFP) fusion restores normal lamellipodial behavior to these cells.¹⁷ Importantly, Arg-YFP colocalizes with F-actin in these protrusive lamellipodial structures.^16,17 In a similar vein, abl^-/-arg^-/- fibroblasts, which have regular smooth peripheries as they adhere to and spread on fibronectin,³⁵ form multiple actin-based filopodial microspikes on fibronectin when Abl is reexpressed in these cells³⁵ (Fig. 2C). Abl localizes to the tips of these filopodia,³⁶ where it may regulate their protrusion, retraction, or adhesion to the extracellular matrix.

These studies demonstrate that Abl and Arg can each coordinate cell protrusions downstream of integrin adhesion receptors. The degree to which Abl and Arg overlap in the regulation of filopodia and lamellipodia is currently unclear. Simultaneous localization of fluorescently-labeled Abl and Arg in fibroblasts should reveal which protrusive structures contain which kinases. The analysis of abl^-/-arg^-/- cells reconstituted with Abl or Arg alone or both kinases should clarify which protrusions depend on which kinases.

Abl Family Kinases Regulate Epithelial Morphogenesis

Genetic studies in flies and mice have revealed a conserved function for Abl family kinases in the regulation of epithelial cell morphogenesis.

The dorsal epidermis in flies is created through the convergent migration and joining of epithelial sheets, a process known as dorsal closure. Cells at the leading edge of the sheet uniformly elongate and assemble a regular actomyosin “purse-string” at their leading edges. The uniform constriction of this purse-string is believed to coordinate the drawing together of epithelial sheets. The Enabled (Ena) protein, a regulator of actin filament length, is enriched at the adherens junctions of these leading edge cells, where it likely helps coordinate the deposition and integrity of the actin purse-string.

Flies that lack both maternal and zygotic sources of fly Abl (abl^MZ mutants) exhibit defects in dorsal epithelial closure.³⁷abl^MZ epithelial cells do not elongate uniformly perpendicular to the closure (Fig. 2D). Ena localization is less uniform in the abl^MZ epithelial cells, with some cells containing larger concentrations of Ena than their neighbors. Cells with more Ena have more F-actin, leading to an uneven organization of the purse string. As a result of these defects, dorsal closure proceeds more slowly and irregularly in abl^MZ embryos and in some embryos, holes in the epidermis remain. Consistent with a possible alteration in adherens junction structure and function, abl^MZ mutants express reduced levels of α-catenin and β-catenin.

Formation of the neural tube, the precursor to the vertebrate brain and spinal cord has many similarities to dorsal closure in flies. The central nervous system begins as a flat sheet of ectoderm along the dorsomedial surface of the developing vertebrate embryo. The cells first undergo microtubule-dependent elongation to form the columnar epithelium of the neural plate.^38-40 Subsequent to this thickening, a contractile actomyosin latticework is formed at the apical surface of these neuroepithelial cells. Contraction of these filaments contributes to the polarized wedge shape that is essential for formation and maintenance of the neural tube.^38,40,41

Abl and Arg are expressed in the mouse neuroepithelium, where they colocalize with the contractile apical actin latticework. abl^-/-arg-/- embryos suffer from severe defects in neural tube formation.^41a Closure of the neural tube is delayed and even incomplete in some embryos. Upon closure, the neural tube of abl^-/- arg^-/- embryos collapses into the central lumen of the neural tube. abl^-/- arg^-/- neuroepithelial cells exhibit patchy disruptions of the apical actin latticework and ectopic actin-rich contractile structures at the basolateral surface. The fact that these defects are never observed in abl^-/- or arg^-/- single mutant embryos, suggests that Abl and Arg play redundant roles neuroepithelial morphogenesis.

Abl Regulates Cellularization in Fly Embryos

abl^MZ mutant flies have a high number of multinucleate cells.⁴² This phenotype can be traced to defects in the formation of the pseudocleavage and cellularization furrows during the late syncytial stages of development.

In the late syncytial stages, nuclei undergo a coordinated localization to the embryo cortex where they continue through 4 coordinated rounds of cell division. During division, the nuclei become separate by an actin-rich pseudocleavage furrow that forms an inverted cup around the metaphase plate. After the final division, a cellularization furrow extends down and engulfs the entire nucleus with membrane.

The formation of pseudocleavage and cellularization furrows is controlled by dynamic rearrangements of the actin cytoskeleton. Actin forms a cap above interphase nuclei, but relocalizes and forms a pseudocleavage furrow during metaphase. Immunohistochemical staining showed that Abl is concentrated at apical junctions and localization extends down into cleavage furrows during these events.⁴³ Some of these pseudocleavage furrows disintegrate in abl mutants, allowing two nuclei to share the same compartment. These defects correlate with increased staining for Ena, Diaphanous, the Arp2/3 complex, and F-actin at the apical surface and reduced F-actin in the pseudocleavage and cellularization furrows. A reduction in Ena levels suppresses the cellularization defects in abl mutants. Together, these studies suggest that Abl acts at the apical surface of the cell to manage Ena localization or activity.

Pathogens Exploit Abl-Regulated Pathways to Infect and Traffic within and between Cells

The ability of Abl family kinases to promote cell protrusions has been hijacked by some viruses and intracellular bacteria as a means to infect and move within and between cells.

The pathogenic bacterium Shigella flexneri infects nonphagocytic cells of the colonic mucosal lining. Contact between Shigella and a target cell induces actin-based protrusive structures that surround and eventually engulf the bacterium.⁴⁴ Abl and Arg localize to bacterial entry sites⁴⁵ and their kinase activities are induced by exposure to Shigella. This induction of kinase activity is critical: abl^-/- arg^-/- cells or wild type cells treated with the Abl/Arg kinase inhibitor STI571 are resistant to Shigella infection. Activation of Abl/Arg by Shigella infection leads to increased phosphorylation of the Crk adaptor protein and is associated with downstream activation of the Rac and Cdc42 GTPases.⁴⁵ Activation of these downstream pathways is likely to underlie the complex actin-based rearrangements required for Shigella internalization.⁴⁵ In support of this, expression of a mutant Crk with a nonphosphorylatable substitution at the Abl/Arg phosphorylation site can inhibit Shigella infection. Once inside cells, Shigella escapes the vacuole and propels itself through the cytoplasm on a “comet tail” of polymerized actin. The IcsA coat protein on Shigella nucleates actin comet formation by recruiting and activating the N-WASP protein to promote Arp2/3 complex-dependent actin polymerization. It is not clear whether Abl family kinases are involved in the formation of the Shigella actin comet tail.

Vaccinia virus, a member of the poxvirus family, also utilizes cellular cytoskeletal systems to traffic within and between cells. Following replication and viral particle assembly in the cytoplasm, some particles become coated with membrane. These intracellular enveloped virions (IEVs) traffic to the cell surface along microtubules using the kinesin motor protein.^46,47 Once at the plasma membrane, the virus induces actin tail formation, which is believed to allow release of cell-associated enveloped virus (CEV) by propelling it away from the cell surface. Src family kinases localize to viral particles where they phosphorylate the vaccinia A36R protein, and recruit the Grb2 and Nck adaptors, N-WASP, and the Arp2/3 complex to promote formation of the actin comet tail.⁴⁸

A recent study also shows that Abl and Arg localize to vaccinia virus comet tails.⁴⁹ Analysis of comet tail formation in mutant cell lines or in cells treated with different kinase inhibitors suggests that comet tail formation may require either Abl family or Src family kinases. Treatment with the selective Abl/Arg inhibitor STI571 (Gleevec) reduced the release of the enveloped virus.⁴⁹ Treatment of animals with STI571 also blocked the spread of vaccinia virus, although it remains possible that the inhibitor may be inhibiting Src or other kinases under the concentrations at which it was used. Nevertheless, this study introduces the possibility of using kinase inhibitors as adjuvants to vaccination to control poxvirus infection.

A Primer: Rho Family GTPases Are Master Regulators of Cytoskeletal Rearrangements

Rho family GTPases, such as Rho, Rac, and Cdc42 act as molecular switches that regulate cytoskeletal rearrangements by cycling between an inactive GDP-bound form and an active GTP-bound form. In their active forms, Rho family GTPases interact with various effectors that regulate actin polymerization, F-actin severing, F-actin bundling, and actomyosin contractility.^50-52 Different Rho family GTPases interface with different effectors to produce different cytoskeletal structures, such as actin stress fibers, filopodia, or lamellipodia.^53-56

Signals originating from cell surface receptors control the activity of Rho family GTPases by acting on two classes of regulatory molecules: guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases by promoting the exchange of GTP for GDP and GTPase-activating proteins (GAPs) that inhibit Rho family GTPases by stimulating them to hydrolyze bound GTP. In some cases, the GEFs and GAPs are controlled directly by cell surface receptors. Alternatively, GEFs and GAPs can be controlled through the activity of a variety of protein kinases and cellular binding partners.

Regulation of the Rac GTPase by Abl Family Kinases

The Rac GTPase is a central regulator of lamellipodial formation and membrane ruffling in response to growth factor receptor signaling.^52,54 Active Rac promotes the recruitment and assembly of complexes containing WASp/WAVE-family proteins which activate the Arp2/3 complex to nucleate new actin filaments.⁵⁷

Several observations in diverse experimental systems have implicated Abl family kinases in the regulation of Rac activity. Rac, Abl, and Arg all become activated in fibroblasts following PDGF treatment and both Rac and Abl activities are essential for PDGF-induced membrane ruffling.^33,58,59 Rac is also activated by Bcr-Abl and expression of a dominant-negative Rac can significantly delay disease onset in a mouse model of Bcr-Abl-induced leukemogenesis.⁶⁰ Similarly, dominant-negative Rac blocks some of the mitogenic effects of the v-Abl oncoprotein in fibroblasts.⁶¹ Rac and Cdc42 are both activated upon cellular exposure to Shigella in wild type cells, but this activation is not observed in abl^-/- arg^-/- cells.⁴⁵ Thus, Abl family kinases control Rac activation in a wide variety of contexts.

Growth-Factor-Induced Rac Activation Requires Abl Phosphorylation of Sos-1

The murine Son-of-Sevenless 1 (Sos-1) can act as a GEF for both Ras and Rac, but acts as a Rac GEF when complexed with the Eps8 and Abi1 proteins following growth factor stimulation.⁶² Exposure of cells to EGF or PDGF leads to increased phosphorylation of Sos-1³⁴ (Fig. 3). This growth factor-induced phosphorylation of Sos-1 is blocked in abl^-/- arg^-/- cells or in wild type cells treated with the Abl/Arg kinase inhibitor STI571. Sos-1 phosphorylation correlates with increased Rac GEF activity and this GEF activity is reduced by treatment with alkaline phosphatase. Growth factor-induced-Rac activation is reduced in abl^-/- arg^-/- cells, but can be increased upon reexpression of Abl. The reduced Rac activation has functional consequences, as abl^-/- and abl^-/- arg^-/- cells exhibit a reduced ruffling response to PDGF.^33,34 These data demonstrate that Abl (and possibly Arg) contribute to Rac activation downstream of growth factor stimulation.

Figure 3

Abl and Arg regulate Rac and Rho downstream of diverse extracellular cues. Left) Binding of platelet-derived growth factor (PDGF) or epithelial growth factor (EGF) to their receptors activates Abl or Arg kinase activities.^, Abl/Arg-mediated phosphorylation (more...)

Interestingly, while Abl phosphorylation stimulates Sos-1 Rac GEF activity, its Ras GEF activity is high irrespective of its phosphorylation status. The basis of this differential requirement for phosphorylation awaits further biochemical studies to elucidate the molecular mechanisms by which Abl phosphorylation regulates Sos-1.

Abl Modulates Rac Activation Downstream of Integrin-Mediated Adhesion

The CAS (Crk-associated substrate) protein becomes heavily phosphorylated in response to integrin-mediated adhesion (see refs. 63 ,64 and references therein). Tyrosine phosphorylation of CAS promotes its assembly into a Rac-activating complex that contains the adaptor protein CrkII (Crk), Elmo, and the Rac GEF Dock180.^63,64 The Crk adaptor contains an SH2 domain followed by two SH3 domains. Crk uses these domains to hold together the Rac activation complex: the Crk SH2 domain binds to phosphorylated CAS,⁶⁵ while the first Crk SH3 domain associates with Dock180.⁶⁶

A number of observations suggest that Abl family kinases regulate the formation of the CAS/Crk/Dock180/Elmo complex. CrkII binds Abl and Arg and serves as their substrate in vitro and in vivo,^9,67,68 with Abl and Arg phosphorylating Crk at a single tyrosine residue (Y221) in a linker region between its two SH3 domains.^9,67 The Crk SH2 domain can bind to its own tyrosine-phosphorylated linker region, and therefore this phosphorylation has been proposed to inhibit Crk binding to other phosphotyrosine-containing proteins, including CAS.⁶⁷ Abl kinase activity levels do negatively correlate with Crk binding to CAS in vivo. For example, whereas abl^-/- arg^-/- fibroblasts have high levels of Crk:CAS containing-complexes, formation of these complexes is reduced in cells reexpressing Abl.⁶⁹

It is difficult to reconcile a simple model in which Abl phosphorylation of Crk inhibits the formation Crk:CAS complexes with the numerous examples (cited above) in which increased Abl kinase activity is associated with Rac activation. The more likely scenario is that phosphorylation of Crk represents an intermediate in the assembly of Crk/CAS/Elmo/Dock180 complexes (Fig. 3). Chodniewicz and Klemke propose a model in which Abl family kinases form complexes with Crk and help relocate it to the plasma membrane.⁶⁴ Once at the membrane the Crk:Abl complex may be dissociated by phosphatases, leaving Crk available to associate with CAS and Dock180. Mutation of tyrosine 221 in Crk to phenylalanine promotes increased complex formation with Abl⁷⁰ and blocks activation of Rac by Abl family kinases.⁴⁵ This residue may play an important role in the dissociating Crk from Abl and promoting its association with CAS and Dock180.

Arg Is Required for Integrin-Dependent Inhibition of Rho

The RhoA (Rho) GTPase promotes the formation of actin stress fibers and focal adhesions in response to growth factor receptor signaling.⁵³ Active Rho activates Rho kinase, thereby promoting actomyosin contractility necessary for focal adhesion and stress fiber formation.⁵²

Arg is particularly abundant in developing brain tissue and analysis of phosphotyrosine-containing proteins during postnatal brain development revealed that the 190 kD GTPase-activating protein for Rho (p190RhoGAP) has reduced tyrosine phosphorylation in arg^-/- brain. Arg phosphorylates p190RhoGAP directly on tyrosine 1105 (Y1105), thereby stimulating its ability to inhibit Rho.⁷¹ Integrin-mediated adhesion to fibronectin promotes p190RhoGAP phosphorylation in wild type fibroblasts. This adhesion-dependent phosphorylation of p190RhoGAP is absent from arg^-/- fibroblasts, but can be restored by reexpression of a functional Arg-YFP fusion protein. Arg-deficient fibroblasts have larger, more numerous focal adhesions and larger stress fibers, a phenotype that appears to result from hyperactive Rho in these cells (A.L. Miller and A.J. Koleske, unpublished data). These observations suggest that Arg acts on p190RhoGAP to inhibit Rho following integrin-mediated cell attachment and spreading (Fig. 3).

Treatment of cultured neurons with the Abl/Arg inhibitor STI571 leads to simplification of neurite structure^35,72,73 that correlates with an increase in Rho activity.⁷² Inhibition of Rho suppresses the effects of STI571 treatment on neurite structure.⁷² This finding supports a model in which Abl family kinases act to inhibit Rho.

Phosphorylation of Y1105 promotes formation of a complex between p190RhoGAP and the 120 kD GTPase activating protein for Ras (p120RasGAP).^74,75 This interaction is mediated in part by binding of one of two SH2 domains in p120RasGAP to the phosphorylated Y1105 in p190RhoGAP.^71,75 Increased p190RhoGAP phosphorylation and increased formation of the p190RhoGAP:p120RasGAP complex correlates with increased disassembly of actin stress fibers in Src-overexpressing or EGF-treated cells.^76,77 These data suggest that Arg-mediated phosphorylation activates p190RhoGAP by promoting its binding to p120RasGAP.

Previous studies had shown that p190RhoGAP is a major cellular target of Src family kinases.⁷⁸ Adhesion-dependent suppression of Rho activity does not occur in fibroblasts that lack the Src, Yes, and Fyn kinases (SYF fibroblasts).⁷⁹ An adhesion-dependent increase in p190RhoGAP phosphorylation is also not observed in these cells. Interestingly, Src family kinases phosphorylate p190RhoGAP at Y1105, the same site targeted by Arg.⁷⁵

Arg and Src family kinases might represent alternative pathways leading from integrin receptors to p190RhoGAP phosphorylation. However, this model would not explain why elimination of either Arg or Src/Fyn/Yes eliminates all adhesion-dependent p190RhoGAP phosphorylation. A more likely scenario is that Arg and Src family kinases are in the same cascade, but only one of the kinases actually phosphorylates p190RhoGAP. Several studies have shown that Src is required for Abl kinase activation upon growth factor receptor stimulation.^33,80 Src family kinases activate Abl family kinases through phosphorylation of an activation loop tyrosine in the Abl/Arg kinase domain.^33,80-82 Arg phosphorylates p190RhoGAP in vitro with a KM of 130 nM. Src can also phosphorylate p190RhoGAP in vitro, but it is unclear how its efficiency compares to that of Arg.⁷⁵ These observations suggest a model in which integrin engagement activates Src to phosphorylate Arg, which, in turn, phosphorylates and activates p190RhoGAP. Alternatively, Arg or Src might be required to set up an appropriate cellular microenvironment to allow phosphorylation of p190RhoGAP by the other kinase.

Abl Interactor (Abi) Proteins Mediate Interactions between Abl Family Kinases and Arp2/3 Regulatory Complexes

The Abi-1 and Abi-2 proteins were identified in two-hybrid screens for proteins that interact with Abl and Arg.^11-13 Abi-1 localizes to filopodial and lamellipodial tips, sites of active actin polymerization.⁸³ Abi-1 and Abi-2 have been identified in protein complexes containing WAVE proteins, which promote Arp2/3 complex-dependent actin polymerization. These complexes also contain the Nap1 and Pir120 proteins.^84-88 Together, these studies raise the possibility that Abi-1 and Abi-2 mediate interactions with Abl family kinases and WAVE proteins. Like Abi-1, Abl and Arg have been localized to sites of actin polymerization at lamellipodial protrusions and filopodial tips. Abl was recently shown to be part of a complex containing WAVE2 and Abi-1.⁸⁹ Adhesion to fibronectin stimulates translocation of these proteins to the cell periphery where they colocalize with F-actin-rich lamellipodia. Activation of Abl kinase activity promotes tyrosine phosphorylation of Abi-1 and WAVE2. Mutation of a single tyrosine residue (Y150) in WAVE2 reduced tyrosine phosphorylation of WAVE2 and assembly of complexes containing WAVE2 and Abi-1. Abl also can stimulate the ability of purified Abi-1/WAVE2 to activate Arp2/3 complex-mediated actin polymerization in vitro. Together, these findings suggest that Abl mediates the formation of an actin polymerization regulatory complex containing in response to integrin-mediated adhesion (Fig. 4A). It is unclear at present whether the Abi-1:Abl:WAVE2 complexes also contain other proteins (e.g., PIR121, Nap1, HSPC300) that have been described in WAVE-containing complexes.

Figure 4

Abl regulates lamellipodial and filopodial formation via distinct mechanisms. A) Promotion of lamellipodial formation by Abl. Integrin-mediated adhesion to the extracellular matrix promotes the assembly of a protein complex containing Abl and Abi-1 and (more...)

The Arg-interacting protein nArgBP2 was also recently shown to bind to WAVE1 and WAVE2.⁹⁰ nArgBP2 might serve as an alternative to Abi proteins to mediate interactions between Abl and WAVE family proteins.

Abl Phosphorylation of Dok Family Proteins Promotes Filopodia Formation

The Downstream of kinase (Dok1) protein was originally identified as a protein that was heavily phosphorylated in Bcr-Abl-transformed cells.^91,92 Subsequent studies have identified 5 family members (Dok1-5), each containing a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) and a C-terminal tail with multiple potential tyrosine phosphorylation sites. Dok1 and Dok2 can serve as essential downstream effectors of Abl in filopodium/actin microspike formation.

Dok1 becomes tyrosine phosphorylated following integrin-mediated adhesion. This phosphorylated Dok1 binds avidly to the Abl SH2 domain.³⁶ Phosphorylation occurs on a single site (Y361) located in the C-terminal tail. In addition to having several potential phosphorylation sites, the Dok2 tail contains a PMMP motif that can interact selectively with the Abl SH3 domain.⁹³ Mutation of this motif abrogates Dok2 association with Abl when the two proteins are coexpressed. Abl expression also promotes Dok2 phosphorylation, which elevates Abl kinase activity, most likely by stabilizing interactions between the two proteins.

Expression of Dok-1 or Dok-2 with Abl leads to increased formation of F-actin microspikes.^36,93 Phosphorylation of Y361 in the Dok1 tail serves as a binding site for the Nck adaptor protein, which can bind to and activate cytoskeletal regulators such as WASp,⁹⁴ N-WASP,⁹⁵ and PAK.⁹⁶ These findings lead to a model in which Abl phosphorylation promotes the assembly of an Abl:Dok1:Nck complex that promotes actin polymerization into microspikes/filopodia (Fig. 4B). In support of this model, Abl and Dok1 both localize to filopodial tips. Cells that lack Dok-1 or both Nck1 and Nck2 form fewer actin-rich filopodia when plated on fibronectin. Ena/VASP family proteins are also found at the filopodial tips where they may regulate the actin filament elongation.⁹⁷

Abl Family Kinases Interact Functionally with Ena/VASP Proteins

Ena/VASP proteins, which include the fly Enabled (Ena) protein, Unc-34/Enabled in worms, and the mammalian Enabled (Mena), VASP (vasodilator-stimulated phosphoprotein), and EVL (Ena/VASP-like protein), regulate the elongation of actin filaments in a number of different cellular structures.⁹⁸ Despite the identification of the enabled gene over 15 years ago as a dosage-dependent modifier of the abl mutant fly phenotypes,⁹⁹ we still do not have a clear mechanistic picture of how Abl family kinases interact at a molecular level with Ena/VASP family proteins.

Genetic studies in flies suggest that Abl may regulate the localization of Ena/VASP family proteins. In the late syncytial divisions and during cellularization in wild type embryos, Ena is distributed at moderate levels at the apical cortex, with weaker levels throughout the cytoplasm.⁴² In abl mutant flies, Ena is strongly enriched at apical surface in association with the increased F-actin structures. This observation suggests that Abl acts to inhibit Ena localization at the apical surface, possibly by interfering with the binding of Ena to one of its partners.

Ena/VASP proteins contain a central proline-rich domain (PRD) that can bind to the Abl SH3 domain. Binding of Abl to the PRD could prevent Ena from binding to other proteins via this domain. Moreover, phosphorylation of specific residues in the PRD of Ena, EVL, and VASP abolishes their ability to bind to SH3-domain-containing proteins.¹⁰⁰ These phosphorylations appear to regulate Ena/VASP protein interactions in response to discrete extracellular cues. For example, adhesion-dependent phosphorylation of VASP by protein kinase A prevents its binding to Abl during initial cell spreading.¹⁰⁰ Abl phosphorylation of Ena in vitro prevents its subsequent binding to the Abl SH3 domain. Vertebrate Abl and fly Abl can promote Mena and Ena phosphorylation respectively when they are coexpressed in cultured cells and this phosphorylation is enhanced by expression of Abi-1 or fly Abi, respectively.^101,102 These data suggest that Abi proteins might stabilize interactions between Ena/VASP proteins and Abl family kinases to allow for Ena/VASP phosphorylation. NABP Future studies should clarify whether this event occurs as part of a response to physiological cues.

Arg Bundles F-Actin and May Form an F-Actin Scaffold to Recruit Cytoskeletal Regulators

Arg contains two distinct F-actin binding domains: a calponin homology (CH) F-actin binding domain at its C-terminus and an I/LWEQ (talin-like) F-actin binding domain located midway between its kinase domain and the CH domain.¹⁶ The region in Abl corresponding to the CH domain in Arg can be separated into two distinct regions that bind to G- and F-actin.¹⁵ An internal domain in Abl corresponding to the I/LWEQ domain can also bind weakly to F-actin (Maithreyi Krishnaswami and A.J.K. unpublished data).

Arg can assemble F-actin into tight bundles in vitro and this activity requires both the I/LWEQ and CH domains.¹⁶ Reconstruction of electron microscopic images of short segments of Arg:F-actin complexes revealed that Arg could bind to F-actin in several modes.¹⁰³ Analysis of Arg mutants lacking either the I/LWEQ or CH domains revealed that the different modes resulted from binding via the different F-actin binding domains. The CH domain bound to subdomain 1 (SD1) of actin and induced a tilt in the actin filament. The I/LWEQ could bind in two modes to either SD1 or SD4. Importantly, only one mode of F-actin binding was found in each Arg:F-actin segment. These data showed that Arg cannot use both domains to bind simultaneously to the same filament. All of the binding modes leave the other domain available to bind an adjacent filament for bundle formation.

Arg binds cooperatively to F-actin in vitro during bundle formation.¹⁶ This property may help concentrate Arg locally within cells, as local Arg binding to F-actin would promote additional binding of Arg to F-actin locally. Electron microscopic structural studies of Arg bound to individual actin filaments show that Arg binding induces structural changes to the actin filament, changing both the structure of actin subunits and their helical pitch.¹⁰³ These structural changes are highly cooperative and propagate along patches of unoccupied actin filament. Importantly, some of the changes in actin filament structure resemble the F-actin conformation found in Arg:F-actin complexes. These findings suggest Arg-induced structural alterations to the actin filament could promote Arg binding to F-actin at sites distal to the initial binding site.

An important unresolved issue is whether Arg organizes F-actin into bundles or higher-order structures within cells. In fibroblasts attaching to fibronectin, Arg localizes to the periphery where it promotes the formation of F-actin-rich protrusive lamellipodial structures. Abl can also bundle F-actin in vitro, and is found in association with F-actin-rich structures in a variety of cell types.^36,104 An Arg C-terminal fragment (Arg688-1182) containing the two F-actin-binding domains is sufficient to form F-actin-rich structures at the cell periphery. These observations suggest that Arg can organize actin filaments into bundles or other higher order structures in vivo. However, while Arg induces highly dynamic F-actin-rich structures that protrude and retract, the F-actin-rich structures induced by Arg688-1182 are largely static. These findings suggest that Arg uses its 688-1182 fragment to localize to the periphery and locally organize F-actin structure, but likely requires other domains to recruit and/or regulate WASp/WAVE proteins and Rho family GTPases to further elaborate these actin-based scaffolds into dynamic structures (Fig. 5). Thus, Arg could simultaneously act as both a building block of an actin bundle scaffold and a regulator of the addition and dynamics of actin filaments arising from this scaffold. Future studies using fluorescence, video and electron microscopy should reveal the cytoskeletal ultrastructure and distribution of regulatory proteins in the Arg-containing protrusive structures.

Figure 5

Arg promotes formation and assembly of an F-actin scaffold at protrusive sites. Integrin-mediated attachment leads to the formation of protrusive Arg- and F-actin-rich protrusive structures at the fibroblast periphery. The following model is proposed (more...)

Abl May Be Subject to Feedback Inhibition by F-Actin

Abl kinase activity is inhibited in fibroblasts that are held in suspension, and is induced upon attachment of cells to fibronectin.^35,105,106 An increased amount of F-actin coimmunoprecipitates with Abl from suspended cells relative to adherent cells. Treatment of cells with Latrunculin A, which reduces cellular F-actin concentration by blocking actin polymerization, leads to a potent induction of Abl kinase activity, even in suspended cells. Together, these observations strongly suggest that Abl kinase activity is influenced by differences in F-actin abundance or conformation in suspended versus adherent cells.

It has been proposed that F-actin inhibits Abl kinase activity by stabilizing an inhibited conformation of the enzyme.¹⁰⁷ Purified F-actin inhibits the ability of Abl to phosphorylate a peptide derived from C-terminal domain (CTD) of RNA polymerase II in vitro.¹⁰⁶ Both the F-actin-binding and SH2 domains of Abl are required for effect. However, F-actin does not inhibit the ability of Abl to phosphorylate its substrate Crk in vitro.¹⁰⁸ One possible explanation for this selective inhibitory effect is that F-actin competes with the CTD for binding to the Abl SH2 or F-actin-binding domains. The CTD binds with low micromolar affinity to an Abl fragment containing the F-actin binding domain.¹⁰⁹ Abl mutants lacking the SH2 or F-actin-binding domains do not bind to F-actin in vitro and therefore would not be inhibited by F-actin. Abl phosphorylation of Crk is not inhibited because Crk does not interact with the SH2 or F-actin binding domains.

A more conventional scenario is that Abl exists in an inactive conformation in unattached cells and integrin-mediated adhesion promotes activation of Abl kinase activity. If this is the case, how does Latrunculin A treatment lead to activation of Abl kinase activity in the absence of adhesion? Latrunculin A treatment of suspended fibroblasts might activate adhesion receptors, mobilize calcium, or activate Rho family GTPases as it has been shown to do in other cell types.^110,111 One or more of these pathways could activate Abl kinase activity.

Abl Family Kinases Mediate Interactions between the F-Actin and Microtubule Networks

Polarized cell migration requires dynamic interactions between the actin and microtubule cytoskeletons.^112,113 MTs assume a polarized orientation in migrating cells, with their plus ends pointed toward the leading edge.^112,113 Microtubule extension into the periphery is required for lamellipodial protrusion at the leading edge.¹¹⁴ Recent studies suggest that Abl family kinases might mediate interactions between F-actin and MTs at the leading edge.

Arg has a microtubule-binding domain located just between its two F-actin-binding domains. ¹⁷ Arg can bind MTs with high affinity and crosslink F-actin bundles to MTs in vitro.¹⁷ These data suggest that Arg might mediate interactions between F-actin and MTs in cells (Fig. 5). In adhering fibroblasts, Arg localizes to the cell periphery and promotes the formation of F-actin-rich lamellipodial protrusions. MTs concentrate and insert into these structures.¹⁷ Elimination of MTs with nocodazole disrupts Arg localization to the periphery, suggesting that Arg:MT interactions help concentrate Arg at protrusive sites. An Arg mutant lacking part of the MT-binding domain does not promote lamellipodial protrusions, even though it can still concentrate at peripheral sites. These data suggest that Arg:MT interactions are essential for Arg to promote lamellipodial protrusion. Arg might serve as part of a peripheral target for MTs.

The microtubule plus end binding protein CLASP binds to a subset of microtubules that interact with the actin-rich periphery in Xenopus growth cones.¹¹⁵ Interestingly, its ortholog Orbit/MAST was identified as a regulator of midline axon guidance in flies. Importantly, genetic studies indicate that Orbit/MAST function is required for axon guidance defects resulting from Abl overexpression in the intersegmental nerve. These data indicate that a critical mediator or MT:F-actin interactions in the growth cone periphery acts downstream of Abl. Future studies should indicate how Abl modulates Orbit/MAST activity during growth cone guidance.