Phosphorylation of c-Abl by protein kinase Pak2 regulates differential binding of ABI2 and CRK

The tyrosine kinase c-Abl is implicated in a variety of cellular processes that are tightly regulated by c-Abl kinase activity and/or by interactions between c-Abl and other signaling molecules. The interaction of c-Abl with the Abl interactor protein Abi2 is shown to be negatively regulated by phosphorylation of serines 637 and 638. These serines are adjacent to the PxxP motif (PTPPKRS637S638SFR) that binds the SH3 domain of Abi. Phosphorylation of the Abl 593-730 fragment by Pak2 dramatically reduces Abi2 binding ( approximately 90%). Mutation of serines 637-639 to alanine (3A) or aspartate (3D) results in an increased tyrosine kinase activity of c-Abl 3D, and a slight reduction of the activity of the 3A mutant, as compared to wild-type (WT) c-Abl. The interaction between Abi2 and c-Abl 3D is inhibited by 80%, as compared to WT c-Abl or c-Abl 3A. This is accompanied by a 2-fold increase in binding of Crk to c-Abl 3D. The data indicate a molecular mechanism whereby phosphorylation of c-Abl by Pak2 inhibits the interaction between the SH3 domain of Abi2 and the PxxP motif of c-Abl. This phosphorylation enhances the association of c-Abl with the substrate Crk and increases c-Abl-mediated phosphorylation of Crk, thus altering the association of Crk with other signaling molecules.     

Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk

Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.     

c-Abl kinase regulates the protein binding activity of c-Crk.

c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation.     

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.     

Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak.

Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Ang1) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Ang1 stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Pak1) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Ang1-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

Consequences of Shb and c-Abl interactions for cell death in response to various stress stimuli

The adaptor protein Shb has previously been shown to regulate apoptosis in response to cytokines and inhibitors of angiogenesis although the mechanisms governing these effects have remained obscure. We currently demonstrate interactions between Shb and c-Abl and that Shb regulates c-Abl kinase activity. The data suggest that c-Abl binds to tyrosine phosphorylated Shb via a concerted effort involving both the c-Abl SH3 and SH2 domains. The biological significance of the Shb/c-Abl interaction was presently tested in overexpression experiments and was found to promote hydrogen peroxide-induced cell death. We also show by Shb knockdown experiments that Shb regulates c-Abl activity and modulates cell death in response to the genotoxic agent cisplatin and the endoplasmic reticulum stress-inducer tunicamycin. The findings are in agreement with the notion of Shb playing a pivotal role in modulating c-Abl pro-apoptotic signaling in response to various stress stimuli.     

Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.

Dok (for downstream of tyrosine kinases) proteins are a newly identified family of docking molecules that are characterized by the presence of an amino-terminal pleckstrin homology (PH) domain, a central putative phosphotyrosine-binding (PTB) domain and numerous potential sites of tyrosine phosphorylation [1] [2] [3] [4] [5] [6]. Here, we explore the potential role of the Dok family member Dok-R (also known as p56(Dok2) or FRIP) in signaling pathways mediated by the epidermal growth factor (EGF) receptor. An intact PTB domain in Dok-R was critical for its association with two PTB-binding consensus sites on the EGF receptor and the PH domain further contributed to stable in vivo binding and tyrosine phosphorylation of Dok-R. Multiple sites on Dok-R were tyrosine-phosphorylated following EGF stimulation; phosphorylated Tyr276 and Tyr304 are proposed to dock the tandem Src homology 2 (SH2) domains of the p21(Ras) GTPase-activating protein rasGAP and Tyr351 mediates an association with the SH2 domain of the adapter protein Nck. Interestingly, we have found that Dok-R could attenuate EGF-stimulated mitogen-activated protein (MAP) kinase activation independently of its association with rasGAP. Together, these results suggest that Dok-R has an important role downstream of growth factor receptors as a potential negative regulator of signal transduction.     

Angiotensin II down-regulates nephrin–Akt signaling and induces podocyte injury: roleof c-Abl

Recent studies have shown that nephrin plays a vital role in angiotensin II (Ang II)–induced podocyte injury and thus contributes to the onset of proteinuria and the progression of renal diseases, but its specific mechanism remains unclear. c-Abl is an SH2/SH3 domain–containing nonreceptor tyrosine kinase that is involved in cell survival and regulation of the cytoskeleton. Phosphorylated nephrin is able to interact with molecules containing SH2/SH3 domains, suggesting that c-Abl may be a downstream molecule of nephrin signaling. Here we report that Ang II–infused rats developed proteinuria and podocyte damage accompanied by nephrin dephosphorylation and minimal interaction between nephrin and c-Abl. In vitro, Ang II induced podocyte injury and nephrin and Akt dephosphorylation, which occurred in tandem with minimal interaction between nephrin and c-Abl. Moreover, Ang II promoted c-Abl phosphorylation and interaction between c-Abl and SH2 domain–containing 5′-inositol phosphatase 2 (SHIP2). c-Abl small interfering RNA (siRNA) and STI571 (c-Abl inhibitor) provided protection against Ang II–induced podocyte injury, suppressed the Ang II-induced c-Abl–SHIP2 interaction and SHIP2 phosphorylation, and maintained a stable level of nephrin phosphorylation. These results indicate that c-Abl is a molecular chaperone of nephrin signaling and the SHIP2-Akt pathway and that the released c-Abl contributes to Ang II–induced podocyte injury.     

Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor-induced cell spreading and morphogenesis

The Crk family of adaptor proteins participate in diverse signaling pathways that regulate growth factor-induced proliferation, anchorage-dependent DNA synthesis, and cytoskeletal reorganization, important for cell adhesion and motility. Using kidney epithelial 293T cells for transient co-transfection studies and the nerve growth factor (NGF)-responsive PC12 cell line as a model system for neuronal morphogenesis, we demonstrate that the non-receptor tyrosine kinase c-Abl is an intermediary for NGF-inducible c-Crk II phosphorylation on the negative regulatory Tyr(222). Transient expression of a c-Crk II Tyr(222) point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr(31) and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr(222) phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl. Interestingly, examination of the early kinetics of NGF stimulation in PC12 cells showed that c-Crk II Tyr(222) phosphorylation preceded paxillin Tyr(31) phosphorylation, followed by a transient initial dissociation of the c-Crk II paxillin complex. PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis. Whereas previous studies have shown that Crk SH2 binding to paxillin is critical for cell adhesion and migration, our data show that the phosphorylation cycle of c-Crk II determines its dynamic interaction with paxillin, thereby regulating turnover of multiprotein complexes, a critical aspect of cytoskeletal plasticity and actin dynamics.     

Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence 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, 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. We show that 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. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.     

Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.     

Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.     

Inhibition of c-Abl tyrosine kinase activity by filamentous actin

The catalytic activity of c-Abl tyrosine kinase is reduced in fibroblasts that are detached from the extracellular matrix. We report here that a deletion of the extreme C terminus of c-Abl (DeltaF-actin c-Abl) can partially restore kinase activity to c-Abl from detached cells. Because the extreme C terminus of c-Abl contains a consensus F-actin binding motif, we investigated the effect of F-actin on c-Abl tyrosine kinase activity. We found that F-actin can inhibit the kinase activity of purified c-Abl protein. Mutations of the extreme C-terminal region of c-Abl disrupted both the binding of c-Abl to F-actin and the inhibition of c-Abl by F-actin. Mutations of the SH3, SH2, and DNA binding domains did not abolish the inhibition of c-Abl kinase by F-actin. Catalytic domain substitutions that affect the regulation of c-Abl by the retinoblastoma protein or the ataxia telangiectasia-mutated kinase also did not abolish the inhibition of c-Abl by F-actin. Interestingly, among these c-Abl mutants, only the DeltaF-actin c-Abl retained kinase activity in detached cells. Taken together, the data suggest that F-actin is an inhibitor of the c-Abl tyrosine kinase and that this inhibition contributes in part to the reduced Abl kinase activity in detached cells.     

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.     

Glutathione peroxidase 1 is regulated by the c-Abl and Arg tyrosine kinases

The c-Abl and Arg tyrosine kinases are activated in the cellular response to oxidative stress. The present studies demonstrate that c-Abl and Arg associate with glutathione peroxidase 1 (GPx1) and that this interaction is regulated by intracellular oxidant levels. The c-Abl and Arg SH3 domains bind directly to a proline-rich site in GPx1 at amino acids 132-145. GPx1 also functions as a substrate for c-Abl- and Arg-mediated phosphorylation on Tyr-96. The results further show that c-Abl and Arg stimulate GPx activity and that these kinases contribute to GPx-mediated protection of cells against oxidative stress. Our findings provide the first evidence that GPx1 is regulated by a signaling pathway that is activated in the oxidative stress response.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

MUC1 oncoprotein blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage

The nonreceptor c-Abl tyrosine kinase binds to cytosolic 14-3-3 proteins and is targeted to the nucleus in the apoptotic response to DNA damage. The MUC1 oncoprotein is overexpressed by most human carcinomas and blocks the induction of apoptosis by genotoxic agents. Using human carcinoma cells with gain and loss of MUC1 function, we show that nuclear targeting of c-Abl by DNA damage is abrogated by a MUC1-dependent mechanism. The results demonstrate that c-Abl phosphorylates MUC1 on Tyr-60 and forms a complex with MUC1 by binding of the c-Abl SH2 domain to the pTyr-60 site. Binding of MUC1 to c-Abl attenuates phosphorylation of c-Abl on Thr-735 and the interaction between c-Abl and cytosolic 14-3-3. We also show that expression of MUC1 with a mutation at Tyr-60 (i) disrupts the interaction between MUC1 and c-Abl, (ii) relieves the MUC1-induced block of c-Abl phosphorylation on Thr-735 and binding to 14-3-3, and (iii) attenuates the MUC1 antiapoptotic function. These findings indicate that MUC1 sequesters c-Abl in the cytoplasm and thereby inhibits apoptosis in the response to genotoxic anticancer agents.     

Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl

Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.