Abl tyrosine kinase


EVOLUTIONARY HOMOLOGS part 1/3

C. elegans Enabled exhibits novel interactions with N-WASP, Abl, and cell-cell junctions

Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells and Drosophila epithelia, but they have only been extensively studied at the leading edges of migratory fibroblasts, where they modulate the protrusion of the leading edge. They act by regulating actin-filament geometry, antagonizing the effects of actin-capping protein. Embryos lacking the C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, the data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function (Sheffield, 2007).

Autoinhibition of c-Abl

Using the specific Abl tyrosine kinase inhibitor STI 571, unphosphorylated murine type IV c-Abl was purified and the kinetic parameters of c-Abl tyrosine kinase activity in a solution was measured with a peptide-based assay. Unphosphorylated c-Abl exhibits substantial peptide kinase activity with K(m) of 204 microm and V(max) of 33 pmol min-1. A transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) has significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl [K(m) = 91 microm, V(max) = 112 pmol min-1]. Autophosphorylation stimulates the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate is dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impairs the activation of wild-type c-Abl by 90% but reduces activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibits the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant is minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245 (Brasher, 2000).

The catalytic activity of the c-Abl tyrosine kinase is tightly regulated by its Src homology 3 (SH3) domain through a complex mechanism that may involve intramolecular binding to Pro242 in the linker region between the SH2 and catalytic domains as well as interactions with a trans-inhibitor. The effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation has been analyzed. Random mutagenesis of SH3 has generated several novel point mutations that dysregulate c-Abl kinase activity in vivo, but the RT loop is insensitive to mutational activation. Activating SH3 mutations abolish binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activate Abl kinase activity in vivo and in vitro but do not impair SH3 ligand-binding. Abl is regulated efficiently when its SH3 domain is replaced with a heterologous SH3 from c-Src that binds a different spectrum of proline-rich ligands, but not by substitution of a modular WW domain with similar ligand-binding specificity. These results suggest that the SH3 domain regulates Abl principally by binding to the atypical intramolecular ligand Pro242 rather than a canonical PxxP ligand. Coordination between the SH3 and SH2 domains mediated by the connector region may be required for regulation of Abl even in the absence of SH2 ligand binding (Brasher, 2001).

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Tyrosine phosphorylation of endogenous c-Abl resuts in a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, phosphorylation and activity are shown to depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. Residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412 have been mutated. These mutations result in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner (Dorey, 2001).

Despite years of investigation, the molecular mechanism responsible for regulation of the c-Abl tyrosine kinase has remained elusive. Inhibition of the catalytic activity of purified c-Abl in vitro is reported, demonstrating that regulation is an intrinsic property of the molecule. The interaction of the N-terminal 80 residues with the rest of the protein mediates autoregulation. This N-terminal 'cap' is required to achieve and maintain inhibition, and its loss turns c-Abl into an oncogenic protein and contributes to deregulation of BCR-Abl (Pluk, 2002).

While Src family kinases have an interaction of their tail with their own SH2 domain that contributes critically to maintenance of the SH3 domain-dependent regulation, c-Abl has an N-terminal cap that serves an analogous function. This cap appears to bind at several regions 'across' the molecule and stabilize the regulated, inhibited conformation. According to the data, the 'KV/LV/LG' motif, which is common to both type 1a and 1b exons and required for binding, must undergo relatively strong interactions with the catalytic domain. The first part of the common second exon does not interact with the catalytic domain but interacts with the SH3 and/or SH2 domains. Because the two regions known to be critical for binding (the cap 1 and cap 6 residues) are spaced differently in the 1a and 1b forms of Abl, some of the 19 additional residues of the type 1b cap may 'loop out' from whatever is the minimal 'bridge' from the SH3 to the catalytic domain. The experiment with the engineered form of c-Abl in which the presence of the N-terminal region is removed in vitro, has shown that the N terminus is required to maintain and not merely to assemble the regulated conformation. This is also confirmed by the ability of cap sequences to inhibit cap-less Abl in vitro. This mechanism may be exploited by cellular proteins that inhibit c-Abl in trans (Pluk, 2002).

The cap is thought to represent the missing link in c-Abl's intramolecular regulation. The first exon region is lacking in all of the different fusion proteins formed with BCR or TEL resulting from chromosomal translocations and also in v-Abl. The dimerization properties of BCR and TEL are thought to induce crossphosphorylation and activation of the catalytic domains by induced proximity and thus represent critical 'gain-of-function' alterations of c-Abl. Moreover, signaling properties in the BCR portion of BCR-Abl are known to be critical for transformation. The data suggest that the absence of the N-terminal cap in BCR-Abl (and in TEL-Abl and v-Abl) represents a 'loss-of-function' alteration that contributes to the acquisition of constitutive tyrosine kinase activity in these oncogenic forms. Thus, the cap of c-Abl may represent what the C-terminal tail represents for c-Src. A crystal structure of c-Abl including the cap will be essential to elucidate the precise molecular mechanism of regulation, and future work will address how the cap may modulate c-Abl differentially in the different splice variants and in the context of cellular signaling networks (Pluk, 2002).

Bcr-Abl is a dysregulated tyrosine kinase whose mechanism of activation is unclear. Like c-Abl, Bcr-Abl is negatively regulated through its SH3 domain. Kinase activity, transformation, and leukemogenesis by Bcr-Abl are all greatly impaired by mutations of the Bcr coiled-coil domain that disrupt oligomerization, but are restored by an SH3 point mutation that blocks ligand binding or a complementary mutation at the intramolecular SH3 binding site defined in c-Abl. Phosphorylation of tyrosines in the activation loop of the catalytic domain and the linker between the SH2 and catalytic domains (SH2-CD linker) is dependent on oligomerization and required for leukemogenesis. These results suggest that Bcr-Abl has a monomeric, unphosphorylated state with the SH3 domain engaged intramolecularly to Pro1124 in the SH2-CD linker, the form that is sensitive to the inhibitor imatinib (STI-571). The sole function of the coiled-coil domain is to disrupt the autoinhibited conformation through oligomerization and intermolecular autophosphorylation (Smith, 2003).

Given these new insights into c-Abl regulation, how is Abl dysregulated upon fusion with Bcr? Relative to c-Abl, the Bcr-Abl fusion protein retains the Abl SH3 domain but lacks Abl first exon sequences and myristoylation and has gained the coiled-coil domain. A simple model suggests that oligomerization through the Bcr coiled-coil domain might constitutively dysregulate the Abl kinase. While deletion of the Bcr coiled-coil domain decreases kinase activity and transformation by Bcr-Abl, the underlying mechanism has not been known. Using novel Bcr coiled-coil mutants, direct evidence has been provided that oligomerization of Bcr-Abl is the important kinase-activating function of the coiled-coil domain. The Bcr-Abl coiled-coil deletion and alanine substitution mutants fail to oligomerize, are defective for transformation of fibroblasts and primary B-lymphoid cells, and are unable to induce CML-like myeloproliferative disease in mice. The low but detectable in vivo tyrosine kinase of monomeric Bcr-Abl may reflect loss of a portion of the Abl N-terminal cap and the myristoyl group. The p210F54P mutant exhibits significant in vivo kinase and cell transforming activity that likely reflects residual oligomerization undetected by a stringent coimmunoprecipitation assay, because the p210F54P/LZA double mutant has low kinase activity and is completely defective for transformation. Phosphorylation of Bcr-Abl at two key regulatory tyrosines is both dependent on oligomerization and required for leukemogenesis, demonstrating that the critical event induced by Bcr-Abl oligomerization is likely to be intermolecular autophosphorylation (Smith, 2003).

Complete deletion of the SH3 domain can restore cell transformation by Bcr-Abl lacking the coiled-coil domain, but the mechanism involved has been unknown. This study demonstrates that in vivo kinase activity, cellular transformation, and leukemogenesis by the Bcr-Abl coiled-coil deletion and alanine substitution mutants are restored by a point mutation (P1013L) in the Abl SH3 domain that disrupts proline-rich ligand binding or by a complementary mutation (P1124L) in the SH2-CD linker region at the site of intramolecular SH3 binding defined in c-Abl. Thus, Bcr-Abl is still autoregulated via its SH3 domain despite the lack of myristoylation, and cannot induce CML unless this inhibition is overcome through oligomerization and transphosphorylation or by SH3 mutation. It has been postulated that oligomerization of Bcr-Abl by the coiled-coil domain might contribute to the pathogenesis of CML through mechanisms other than direct effects on Bcr-Abl kinase activity, such as crosslinking of F-actin microfilaments. However, the results demonstrate that Bcr-Abl can induce CML-like disease independent of oligomerization when released from its autoinhibited state by other means (Smith, 2003).

Taken together, these observations suggest that an elegant and simple mechanism governs Bcr-Abl catalytic activity. The results argue that Bcr-Abl can assume an inactive state where the enzyme is monomeric and unphosphorylated. The primary consequence of oligomerization of Bcr-Abl is intermolecular autophosphorylation at Tyr1294 in the activation loop of the catalytic domain. As in c-Abl, Tyr1294 phosphorylation may lead to secondary phosphorylation events, including phosphorylation of Tyr1127, the homolog of c-Abl Tyr245, which results in displacement of the SH3 domain from the SH2-CD linker. The order of phosphorylation may not be obligatory, since autophosphorylation of the linker tyrosine in both c-Abl and Bcr-Abl can occur when the activation loop tyrosine is mutated. Enzymological studies of purified Bcr-Abl proteins should be helpful in confirming this model. While there are additional tyrosine phosphorylation sites in Bcr-Abl and c-Abl, the results suggest that Tyr1294 and Tyr1127 are the major sites that influence the regulation of catalytic activity in both enzymes. Other phosphorylation sites may contribute to leukemogenesis by Bcr-Abl independent of any role in autoregulation, as is the case with Tyr177, which forms a binding site for the SH2 domain of Grb2 and is required for induction of CML-like leukemia by Bcr-Abl (Smith, 2003).

The c-Abl tyrosine kinase is inhibited by mechanisms that are poorly understood. Disruption of these mechanisms in the Bcr-Abl oncoprotein leads to several forms of human leukemia. Like Src kinases, c-Abl 1b is activated by phosphotyrosine ligands. Ligand-activated c-Abl is particularly sensitive to the anti-cancer drug STI-571/Gleevec/imatinib (STI-571). The SH2 domain-phosphorylated tail interaction in Src kinases is functionally replaced in c-Abl by an intramolecular engagement of the N-terminal myristoyl modification with the kinase domain. Functional studies coupled with structural analysis define a myristoyl/phosphotyrosine switch in c-Abl that regulates docking and accessibility of the SH2 domain. This mechanism offers an explanation for the observed cellular activation of c-Abl by tyrosine-phosphorylated proteins, the intracellular mobility of c-Abl, and it provides new insights into the mechanism of action of STI-571 (Hantschel, 2003).

Starting from the regulated conformation of the kinase, c-Abl can be activated by either SH3 or SH2 domain ligands, leading to displacement of the inhibitory clamp from the backside of the kinase domain. Myristoyl displacement by a yet unidentified class of fatty-acid binding proteins might lead to a concomitant disruption of the SH2-kinase domain interface. These mechanisms might lead to a fully activated form of the kinase, which can be further stabilized, as well as initiated, by phosphorylation of the activation loop tyrosine Tyr-412 and/or Tyr-245 in the SH2-kinase linker. Once phosphorylated, c-Abl can reassume its regulated state upon the action of cellular phosphatases (Hantschel, 2003 and references therein).

The cap region can partly restore inhibition of a dimerization-defective form of the Bcr-Abl fusion protein. This implies that regulatory constraints and activation modes may be operational within Bcr-Abl. Specifically there are four potential activation mechanisms for c-Abl 1b: SH3 domain-dependent activation, SH2 domain-dependent activation, activation by phosphorylation on Tyr-412 (activation loop) or Tyr-245 (SH2-kinase linker), and activation by myristoyl displacement. This view has now been confirmed by an elegant genetic analysis revealing that mutations in all surfaces proposed to be participating in c-Abl regulation, including the cap, confer STI-571 resistance to Bcr-Abl. Residual regulatory constraints must therefore be operational within Bcr-Abl despite the lack of a myristoyl group and in competition with the activation caused by BCR-driven dimerization (Hantschel, 2003 and references therein).

The crystal structure of the myristoylated form of c-Abl shows regions of electron density in the vicinity of the SH3 domain N terminus likely to represent interaction sites of the C-terminal part of the cap (functionally identified Lys-70 and Leu-73). The previously documented interaction of a GST-Cap fusion protein with the SH3-SH2 domain must mainly occur in this region. Phosphotyrosine-ligand activated c-Abl, in which the conformational restraints conferred by the SH3/SH2 clamp are not operational, is particularly sensitive to inhibition by STI-571. This suggests that 'breathing' of the N-terminal and C-terminal lobes is an important parameter for the selectivity of protein kinase inhibitors, which may need to be 'swallowed'. These observations offer a potential explanation for the relative safety of STI-571 in treatment of leukemia patients. Since the developmental effects of loss of function of the ABL1 or/and ABL2 genes in the mouse are quite severe, one could have anticipated toxicity problems through inhibition of the cellular form of the enzyme (Hantschel, 2003 and references therein).

The myristoyl pocket appears to be quite specific for c-Abl and Arg since the corresponding space in Src kinases is filled with bulky side chains. It is possible that the myristoyl binding pocket offers new opportunities for Bcr-Abl inhibitors since molecules that bind to it might be able to stabilize the inactive and assembled conformation of the enzyme (Hantschel, 2003).

c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571) to inhibit the catalytic activity of Abl, but not that of c-Src (Nagar, 2003).

Mutation of vertebrate ABL

Mice homozygous for an Abl m1 mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. The immune system shows major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments. (Schwartzberg, 1991). Most mice homozygous for the c-abl mutation become runted and die 1 to 2 weeks after birth. In addition, many show thymic and splenic atrophy and a T and B cell lymphopenia (Tybulewicz, 1991).

Activation of the c-Abl proto-oncogene occurs in Abelson murine leukemia virus, Hardy-Zuckerman-2 feline sarcoma virus, and during the Philadelphia chromosomal translocation that generates the BCR/ABL fusion gene. The three genes exhibit varying degrees of transforming activity; the two viral genes transform NIH-3T3 cells in vitro, whereas the BCR/ABL gene is incapable of transforming these cells. To determine whether genetic alterations can enhance the transforming potential of the BCR/ABL gene, genetic selection techniques were employed which led to the isolation of a mutant form of the BCR/ABL gene with high levels of fibroblastic transforming activity. Molecular analysis of this clone shows that it suffers a deletion of 3' ABL sequences and their replacement with a cellular sequence of unknown origin, termed X. This tripartite gene is capable of inducing 35 foci/10 ng of DNA. Deletion of 3' ABL sequences analogous to those seen in the activated BCR/ABL protein without the addition of X yields 5 foci/100 ng of DNA. These results suggest that carboxyl-terminal truncations unmask the fibroblastic transforming activity of the BCR/ABL gene product and the addition of X sequences dramatically enhances this transforming potential, indicating a dominant contribution by the X reading frame (Shore, 1994).

Overexpression of c-Abl tyrosine kinase can be growth inhibitory in certain fibroblast cell lines. Using a series of conditional chimeras between Abl and Src, the Abl protein was dissected to determine which domains are required for this function. Growth inhibition, unlike transformation by oncogenic forms of Abl, is dependent on the presence of the cognate SH2 and tyrosine kinase domains. Since growth inhibition correlates with low tyrosine kinase activity, it may involve highly specific interactions of target proteins with both domains without the processivity of phosphorylation associated with oncogenic Abl (Mattioni, 1996).

Two loss-of-function point mutations were introduced from highly conserved regions of the src homology 3 (SH3) domains of the Caenorhabditis elegans sem-5 gene into the SH3 domain of the murine type IV c-Abl tyrosine kinase proto-oncogene. One of the mutations, P131L, activates Abl to transform fibroblasts while the other, G128R, does not. When combined with independent activating mutations in the c-Abl kinase domain or NH2-terminus, the G128R mutation blocks transformation by the double mutant, suggesting that the G128R mutant is unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c-Abl protein, is localized to the nucleus and actin cytoskeleton and has normal tyrosine kinase activity in vitro, while the transforming c-Abl P131L protein is localized exclusively to the cytoplasm and exhibits decreased in vitro kinase activity. The wild type murine Abl SH3 domain binds to two proteins containing proline-rich motifs with dissociation constants of 0.2 and 17 microM; the G128R mutant binds with 50-fold lower affinity, and no binding is detected by the P131L mutant. Both mutations completely abolish binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay. These results suggest that the transforming activity of Abl is regulated in vivo by an inhibitor protein which associates with the SH3 domain via a proline-rich sequence (Van Etten, 1995).

Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. The Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg-/- fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin-rich cell protrusions. Arg requires both its F-actin-binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg-/- fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts.

Proteins that interact with ABL and ABL targets

A mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, is an adaptor molecule functioning in neural development. mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks (Howell, 1997).

Activation of phosphatidylinositol (PI) 3-kinase by growth factors results in phosphorylation of phosphatidylinositol lipids at the D3 position. Although PI 3-kinase is essential to cell survival, little is known about mechanisms that negatively regulate this activity. The c-Abl tyrosine kinase interacts directly with the p85 subunit of PI 3-kinase. Activation of c-Abl by ionizing radiation exposure is associated with c-Abl-dependent phosphorylation of PI 3-kinase. Phosphorylation of p85 by c-Abl inhibits PI 3-kinase activity in vitro and in irradiated cells. These findings indicate that c-Abl negatively regulates PI 3-kinase in the stress response to DNA damage (Yuan, 1997b).

The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase. The kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain. Point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, a cDNA library was screened by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. A novel protein was identified, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrate that in the presence of AAP1, c-ABL is inhibited from phosphorylating either glutathione S-transferase-CRK or enolase. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. It is concluded that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein. It is proposed that AAP1 functions as a trans regulator of c-ABL kinase (Zhu, 1996).

A novel cellular protein, Abl-interactor-1 (Abi-1), which specifically interacts with the carboxy-terminal region of Abl oncoproteins, has been identified in a mouse leukemia cell line. The protein exhibits sequence similarity to homeotic genes, contains several polyproline stretches, and includes an src homology 3 (SH3) domain at its carboxyl-most terminus that is required for binding to Abl proteins. The abi-1 gene has been mapped to mouse chromosome 2 and is genetically closely linked to the c-abl locus. The gene is widely expressed in the mouse, with highest levels of mRNA found in the bone marrow, spleen, brain, and testes. The Abi-1 protein coimmunoprecipitates with v-Abl and serves as a substrate for kinase activity. When overexpressed in NIH-3T3 cells, abi-1 potently suppresses the transforming activity of Abelson leukemia virus expressing the full-length p160v-abl kinase but does not affect the transforming activity of viruses expressing a truncated p90v-abl or v-src kinases. It is suggested that the Abi-1 protein may serve as a regulator of Abl function in transformation or in signal transduction (Shi, 1995).

Although numerous targets for the Abl kinases have been identified, only a few of these have been shown to be important in modulation of the Abl-transforming potential. A family of Abl-interactor (Abi) proteins have been identified that bind specifically to both the SH3 and carboxy-terminal proline-rich sequences of Abl. Two distinct, yet highly related genes, abi-1 and abi-2, have been identified and cloned. The corresponding protein products share overall 69% identity with the greatest homology observed in the amino-terminal homeobox-like domain, proline-rich sequences, and the carboxy-terminal SH3 domain. The Abi proteins are substrates of the Abl kinases. Significantly, Abi proteins antagonize the oncogenic activity of Abl in fibroblasts. Overexpression of Abi-1 potently suppresses the transforming activity of viral Abl (v-Abl) in NIH-3T3 fibroblasts. Coexpression of a truncated form of Abi-2 with c-Abl activates the oncogenic potential of c-Abl. These and other data suggest that the full-length Abi proteins may function as growth inhibitors in mammalian cells. The destruction of the Abi proteins requires tyrosine kinase activity and is dependent on the ubiquitin-proteasome pathway. Degradation of the Abi proteins occurs through a Ras-independent pathway. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemias. These findings suggest that loss of Abi proteins may be a component in the progression of Bcr-Abl-positive leukemias and identify a novel pathway linking activated nonreceptor protein tyrosine kinases to the destruction of specific target proteins through the ubiquitin-proteasome pathway (Dai, 1998).

p62dok is a docking protein and a target of the ABL tyrosine kinase. p62dok is constitutively tyrosine-phosphorylated in chronic myelogenous leukemia progenitor cells and is associated with ras GTPase-activating protein (GAP)-associated protein (See Drosophila Ras1). Association of p62dok with GAP correlates with p62dok's tyrosine phosphorylation. p62dok is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases. Given the important signaling role that tyrosine kinases play in hematopoietic control, it is not surprising that the appearance of a novel tyrosine kinase activity within a single, primitive erythroid progenitor cell would perturb the intracellular signaling cascades that ensure orderly hematopoiesis (Carpino, 1997).

The adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase (see below, De Jong, 1997), and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Philadelphia chromosome-positive patient material, containing an active Bcr/Abl protein. The 120 kDa protein is Cbl, originally discovered as an oncogene that induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl (de Jong, 1995).

The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. CRKL phosphorylation by the Abl kinase is limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yields a mutant CRKL that can not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F does not transform NIH3T3 cells, while the Y207F mutation eliminates tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL does not enhance or decrease association with various target signaling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation (de Jong, 1997).

The Philadelphia chromosome, detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR-encoded sequences upstream of exon 2 of c-ABL. The BCR-ABL fusion creates a gene whose protein product, p210BCR-ABL, has been implicated as the cause of the disease. Although ABL kinase activity has been shown to be required for the transforming abilities of BCR-ABL and numerous substrates of the BCR-ABL tyrosine kinase have been identified, the requirement of most of these substrates for the transforming function of BCR-ABL is unknown. There is a direct binding site of the c-CBL proto-oncogene to the SH2 domain of BCR-ABL. This interaction only occurs under conditions where c-CBL is tyrosine-phosphorylated. Despite the direct interaction of c-CBL with the SH2 domain of BCR-ABL, the deletion of the SH2 domain of BCR-ABL does not result in an alteration in the complex formation of BCR-ABL and c-CBL. This suggests that another site of direct interaction between c-CBL and BCR-ABL exists or that another protein mediates an indirect interaction of c-CBL and BCR-ABL. Since CRKL, an SH2, SH3 domain-containing adapter protein is known to bind directly to BCR-ABL and also binds to tyrosine-phosphorylated c-CBL, the ability of CRKL to mediate a complex between c-CBL and BCR-ABL was examined. Such a three way complex is confirmed (Bhat, 1997).

In primary leukemic neutrophils from patients with chronic myelogenous leukemia (CML), the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein that has an overall homology of 60% to CRK, the human homolog of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL is tyrosine phosphorylated, while CRK is not. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitate with CRKL: C3G, SOS and c-ABL. Each of these proteins is found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins do not change, with the exception that BCR/ABL now coprecipitate with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bind to a new complex of proteins of approximate molecular weight 105-120 kDa. The major protein in this complex is p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes that potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120CBL (Uemura, 1997).

The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. The kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. Both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo (Shishido, 2000).

Cbl can form a closed structure that prevents the binding of Src to Cbl. The binding of Abl to Cbl is considered to trigger the structural changes in Cbl. In the case of other proteins that have a closed structure such as Src or Crk, the deletions of the C-terminal regions give rise to the formation of oncogenic proteins v-Src and v-Crk. Similarly, v-Cbl has a deletion of the C-terminal region of Cbl, including the proline-rich region of Cbl. These results suggest that the oncogenic activity residing in the N-terminal region of Cbl can be released because of a disruption of a closed structure of Cbl. In addition, Cbl has another oncogenic form, 70Z Cbl, which contains a 17-aa deletion between the N-terminal and the C-terminal regions. 70Z Cbl constitutively binds to Src although WT Cbl does not. This result also suggests that the constitutive conformational changes that open the structure of Cbl contribute to its oncogenic activation. The Cbl ring finger domain exhibits the ubiquitin ligase activity. The structural transition of Cbl induced by Abl might regulate the ligase activity because the ring finger domain is localized right next to the Src binding proline-rich region (Shishido, 2000).

In summary SrcKD directly inhibits Cbl phosphorylation by the Abl kinase. The analysis of mutants suggests that the complex of SrcKD and Cbl acts as an inhibitor of Abl. Although the physiological consequence of the interaction and the regulation of Abl, Cbl, and kinase-inactive form of WT Src is still unclear at the moment, it is possible that these interactions are important for the cytoskeleton reorganization. The kinase-activated Src activates Abl and the kinase-deficient Src reduces this activation in response to platelet-derived growth factor-regulating membrane ruffling. SrcKD regulates the cytoskeleton reorganization in osteoclasts, and Cbl has been found to act downstream of Src in these cells. The mechanism of Abl inhibition by SrcKD would not be limited to the inhibition of the WT Src kinase but would rather ensue from direct inhibition of the Abl kinase by SrcKD. Thus, this study suggests caution in interpreting data using a kinase-deficient molecule as a dominant negative to study the kinase activity (Shishido, 2000 and references therein).

What is the effect of the v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) on the Jak-STAT pathway of cytokine signal transduction? In murine pre-B lymphocytes transformed with A-MuLV, the Janus kinases (Jaks) Jak1 and Jak3 (see Drosophila Hopscotch) exhibit constitutive tyrosine kinase activity, and the STAT proteins (signal transducers and activators of transcription) (see Drosophila Marelle) normally activated by interleukin-4 and interleukin-7 are tyrosine-phosphorylated in the absence of these cytokines. Coimmunoprecipitation experiments reveal that in these cells v-Abl is physically associated with Jak1 and Jak3. Inactivation of v-Abl tyrosine kinase in a pre-B cell line transformed with a temperature-sensitive mutant of v-abl results in abrogation of constitutive Jak-STAT signaling. A direct link may exist between transformation by v-abl and cytokine signal transduction (Danial, 1995).

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl-Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. It is proposed that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene (Danial, 1998).

Biochemical and genetic evidence suggests that the tyrosine kinase activity of c-Abl is tightly regulated in vivo by a cellular factor binding to the Src homology 3 (SH3) domain of Abl. The yeast two-hybrid system was used to identify a gene, PAG, whose protein product (Pag) interacts specifically with the Abl SH3 domain. Pag, also known as macrophage 23-kD stress protein (MSP23), is a member of a novel family of proteins with antioxidant activity implicated in the cellular response to oxidative stress and in control of cell proliferation and differentiation. In a co-expression assay, Pag associates with c-Abl in vivo and inhibits tyrosine phosphorylation induced by overexpression of c-Abl. Inhibition requires the Abl SH3 and kinase domains and is not observed with other Abl SH3-binding proteins. Expression of Pag also inhibits the in vitro kinase activity of c-Abl, but not SH3-mutated Abl or v-Abl. When transfected in NIH-3T3 cells, Pag is localized to the nucleus and cytoplasm and rescues the cytostatic effect induced by c-Abl. These observations suggest Pag is a physiological inhibitor of c-Abl in vivo (Wen, 1997).

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, the SH3 domain of c-Abl was identified as interacting with the proline-rich p12 domain of Pr60gag. The two proteins associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells leads to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells (Dupraz, 1997).

The products of the human Philadelphia chromosome translocation, P210 and P190(BCR/ABL), are cytoplasmic protein tyrosine kinases that share the ability to transform hematopoietic cytokine-dependent cell lines to cytokine independence but differ in the spectrum of leukemia induced in vivo. STAT5 and, to a lesser extent, STATs 1 and 3 are constitutively activated by tyrosine phosphorylation and induction of DNA binding activity in both P210 and P190(BCR/ABL)-transformed cells, but P190 differs in that it also prominently activates STAT6. There is low level tyrosine phosphorylation of JAKs 1, 2, and 3 in Bcr/Abl-transformed cells, but no detectable complex formation with Bcr/Abl; activation of STAT5 by P210 is not blocked by two different dominant-negative JAK mutants. These results suggest that P210 and P190(BCR/ABL) directly activate specific STAT family members and may help explain their overlapping yet distinct roles in leukemogenesis (Ilaria, 1996).

Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. hTERT is shown to associate directly with the c-Abl protein tyrosine kinase. c-Abl phosphorylates hTERT and inhibits hTERT activity. Exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. It is concluded that the ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. The finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function (Kharbanda, 2000).

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables (Cdk5 and Abl enzyme substrate), a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Cables displays little sequence homology to other known proteins in the databases. It does, however, show weak homology to cyclin A and weaker homology to cyclin C over an ~200 amino acid stretch in the C-terminal third of the protein, which may be the Cdk-interacting region. Cables also contains six PXXP motifs, defined as the minimal consensus for SH3 domain binding, and two tyrosine-based sorting motifs (YXXLE), which have been implicated in axonal growth cone sorting. It contains three serine proline/threonine proline minimal Cdk phosphorylation sites and at least one potential c-Abl phosphorylation site (YXXP). Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibits neurite outgrowth. Furthermore, expression of active Abl results in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system (Zukerberg, 2000).

These data suggest that Cables serves as an adaptor molecule, facilitating Cdk5 tyrosine phosphorylation and regulation by c-Abl. Phosphorylation of key substrates involved in actin and microtubule dynamics by active Cdk5 is likely to contribute to its role in neuronal migration and neurite outgrowth. Furthermore, Cdk5 has been shown to downregulate N-cadherin-mediated cell adhesion. Data presented in this communication suggest that Cables mediates an interaction between c-Abl and Cdk5, and may positively affect brain development and neurite outgrowth by enhancing Cdk5 tyrosine phosphorylation and upregulation of kinase activity. Cables may also mediate an interaction between Cdk5 and mDab1 by binding to both Cdk5 and c-Abl (Zukerberg, 2000).

A search for c-Abl interacting proteins resulted in the recovery of PSTPIP1, originally identified as a binding protein of the PEST-type protein tyrosine phosphatases (PTP). The PEST nomenclature for PTPs, an only partially characterized family of proteins, is a misnomer because they do not have short half-lives. PSTPIP1 is a cytoskeletal protein previously identified as a binding partner of the PEST-type tyrosine phosphatase. PSTPIP1 contains an SH3 domain at its carboxyl terminus and has a significant homology to Schizosaccharomyces pombe CDC15p, a key element in the reorganization of F actin during mitosis. PSTPIP1 colocalizes with cortical actin cytoskeleton, stress fiber, lamellipodia, and the cytokinetic cleavage furrow. Transiently expressed PSTPIP1 is also found at focal adhesion contacts. Overexpression of PSTPIP1 in 3T3 cells results in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. PSTPIP1 is phosphorylated by c-Abl, and growth factor-induced PSTPIP1 phosphorylation is diminished in Abl null fibroblasts. PSTPIP1 is able to bridge c-Abl to the PEST-type PTPs. Several experiments suggest that the PEST-type PTPs negatively regulate c-Abl activity: c-Abl is hyperphosphorylated in PTP-PEST-deficient cells; disruption of the c-Abl-PSTPIP1-PEST-type PTP ternary complex by overexpression of PSTPIP1 mutants increases c-Abl phosphotyrosine content; and PDGF-induced c-Abl kinase activation is prolonged in PTP-PEST-deficient cells. Dephosphorylation of c-Abl by PEST-type PTP represents a novel mechanism by which c-Abl activity is regulated. These findings suggest that PSTPIP1 acts as an adaptor protein by recruiting PEST-type PTP to c-Abl. Although PTP-PEST does not interact directly with c-Abl, it is able to form a complex with c-Abl through the adaptor protein PSTPIP1, and thus the proline-rich sequence at the C terminus of PEST-type PTP is important for targeting c-Abl. This study illustrates how complex formation contributes to the substrate selectivity of PTP. PSTPIP1 can also couple PEST-type PTP to other substrates besides c-Abl. CD2BP1, a human homolog of PSTPIP1, binds to the intracellular tail of CD2 and down-regulates CD2-triggered cell adhesion by coupling PTP-PEST to CD2 (Cong, 2000).

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. WAVE-1 (Drosophila homolog: SCAR) has been identified in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin because the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, a previously unrecognized function is proposed for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl (Westphal, 2000).

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia, these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins (Drosophila homolog: Abelson Interacting Protein), targets of Abl tyrosine kinases, have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation. The unique localization of Abi proteins in living, motile cells is described in this study. Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. The targeting domain is identified as the homologous N terminus of these two proteins. These findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility (Stradal, 2001).

The cytosolic domain of the ß-amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of these is Mena, which binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologs of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr682 of APP695 is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling (Zambrano, 2001).

It is worth noting that the Tyr682 of human APP695 and the YENP motif are both conserved among all the known APPs in primates, rodents, Drosophila, and Caenorhabditis and are present also in the APP-related proteins APLP1 and APLP2. Considering that the overall sequence identity between Drosophila APP (Appl) and the mammalian APPs is less than 30%, the 100% conservation of the cytosolic motif containing the phosphorylated tyrosine suggests that it plays a key functional role. This means that the understanding of the molecular basis of the different phenotypes observed in insects bearing mutations of Drosophila Abl (DAbl) and/or disabled and/or enabled should also take into account the involvement of APP. Appl null flies show behavioral defects that are rescued by human APP, and the possible correlation with the defects caused by DAbl, disabled, and enabled gene mutations is not apparent. However, one could gain better insight by the analysis of the phenotypes of insects bearing combined mutations of Appl with the other three genes. For example, the effects of disabled gene mutation on the Abl -/- flies also could be the consequence of the direct interaction of these two proteins with APP, whereas the amelioration observed in Drosophila Abl-/-;disabled-/- following the mutation of the enabled gene could be also based on the competition between the enabled and DAbl gene products for the binding to Appl through Drosophila Fe65 (Zambrano, 2001).

Although the WW domain of Fe65 interacts in vitro with both c-Abl and Abl-PP, only the complexes between Fe65 and the active form of Abl, and not those with the wild type c-Abl, were found in cell extracts. This effect could be due to a lower amount of c-Abl than Abl-PP available for the formation of the in vivo complexes; or it could be due to a low affinity of c-Abl for the WW domain of Fe65 so that, in vivo, it cannot form a significant number of complexes with Fe65 because of the competition of the other ligands of the WW domain of this protein. Furthermore, active Abl probably has a different conformation from that of c-Abl, thus acquiring a higher affinity for the WW domain. On the contrary, the APP-Abl direct interaction probably requires an active Abl, because the binding is based on a pTyr-SH2 interaction (Zambrano, 2001).

It has been hypothesized often that APP could have some role in signaling, and in a recent review article, Bothwell and Giniger (2000) suggested the possibility that intracellular signaling could be involved in the development of AD. Their hypothesis takes into account the numerous reports on various proteins that could be involved in the pathogenesis of AD and suggests a role for c-Abl as a modulator of APP biology. The results presented here support their hypothesis. A point that deserves attention concerns the possible involvement of p73 in the molecular machinery under examination. In fact, this protein is a key regulator of apoptosis that binds to and is activated by Abl as a response to DNA damage. An isoform of p73 functions as an anti-apoptotic protein in developing neurons, and the role of its phosphorylation by Abl has not been addressed. The finding that active Abl binds to APP suggests an examination of the possible regulatory effects of this binding on the p73 phosphorylation by Abl and the consequences on this regulation of the enhanced APP proteolytic processing characteristic of AD (Zambrano, 2001).

C-Abl is a nonreceptor tyrosine kinase that is tightly regulated in the cell. Genetic data derived from studies in flies and mice strongly support a role for Abl kinases in the regulation of the cytoskeleton. C-Abl can be activated by several stimuli, including oxidative stress, DNA damage, integrin engagement, growth factors, and Src family kinases. Structural alterations elicit constitutive activation of the c-Abl tyrosine kinase, leading to oncogenic transformation. While the mechanisms that activate c-Abl are beginning to be elucidated, little is known regarding the mechanisms that downregulate activated c-Abl. Activated c-Abl is downregulated by the ubiquitin-dependent degradation pathway. Activated forms of c-Abl are more unstable than wild-type and kinase-inactive forms. Moreover, inhibition of the 26S proteasome leads to increased c-Abl levels in vitro and in cells, and activated c-Abl proteins are ubiquitinated in vivo. Significantly, inhibition of the 26S proteasome in fibroblasts increases the levels of tyrosine-phosphorylated, endogenous c-Abl. These data suggest a novel mechanism for irreversible downregulation of activated c-Abl, which is critical to prevent the deleterious consequences of c-Abl hyperactivation in mitogenic and cytoskeletal pathways (Echarri, 2001).

STAT5 is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. The BCR/ABL SH3 and SH2 domains interact with hematopoietic cell kinase (Hck), leading to the stimulation of Hck catalytic activity. Active Hck phosphorylates STAT5B on Tyr699, which represents an essential step in STAT5B stimulation. Moreover, a kinase-dead Hck mutant and Hck inhibitor PP2 abrogates BCR/ABL-dependent activation of STAT5 and elevation of expression of STAT5 downstream effectors A1 and pim-1. These data identify a novel BCR/ABL-Hck-STAT5 signaling pathway, which plays an important role in BCR/ABL-mediated transformation of myeloid cells (Klejman, 2002).

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. This study identifies a constitutive BRCA1-c-Abl complex and evidence is provided for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner (see Drosophila mei-41), which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. The C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. A model is suggested in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity (Foray, 2002).

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase [p70(S6k)] and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation (Kumar, 2000).

Abl family kinases, which include the mammalian Abl and Arg (Abl-related gene) kinases, regulate neuronal morphogenesis in developing metazoa. Activation of Abl kinase activity directs changes in actin-dependent processes such as membrane ruffling, filopodial protrusion, and cell motility. However, the mechanisms by which increased Abl or Arg kinase activity promote cytoskeletal rearrangements are unclear. Evidence is provided that the Rho inhibitor p190RhoGAP (GTPase-activating protein) is an Arg substrate in the postnatal mouse brain. p190RhoGAP has reduced phosphotyrosine content in postnatal arg-/- mouse brain extracts relative to wild-type extracts. In addition, the adhesion-dependent stimulation of p190RhoGAP phosphorylation observed in wild-type cells is not observed in arg-/- fibroblasts and neurons. Arg can phosphorylate p190RhoGAP in vitro and in vivo on tyrosine (Y) 1105. Arg can stimulate p190RhoGAP to inhibit Rho and Arg-mediated phosphorylation is required for this stimulation. Phosphorylation by Arg also promotes p190RhoGAP's association with p120RasGAP and stimulates p190RhoGAP's ability to induce neuritogenesis in neuroblastoma cells. These results demonstrate that p190RhoGAP is an Arg substrate in the developing brain and suggest that Arg mediates the adhesion-dependent regulation of neuronal morphogenesis in the postnatal brain by phosphorylating p190RhoGAP (Hernández, 2004).

c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family, has been identified. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays reveal that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 has a synergistic effect on apoptosis induction and leads to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappear from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role (Li, 2002).

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. It was previously shown that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) part 2/3 ! part 3/3


Abl tyrosine kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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