c-Abl downregulates the slow phase of double-strand break repair

c-Abl tyrosine kinase is activated by agents that induce double-strand DNA breaks (DSBs) and interacts with key components of the DNA damage response and of the DSB repair machinery. However, the functional significance of c-Abl in these processes, remained unclear. In this study, we demonstrate, using comet assay and pulsed-field gel electrophoresis, that c-Abl inhibited the repair of DSBs induced by ionizing radiation, particularly during the second and slow phase of DSB repair. Pharmacological inhibition of c-Abl and c-Abl depletion by siRNA-mediated knockdown resulted in higher DSB rejoining. c-Abl null MEFs exhibited higher DSB rejoining compared with cells reconstituted for c-Abl expression. Abrogation of c-Abl kinase activation resulted in higher H2AX phosphorylation levels and higher numbers of post-irradiation γH2AX foci, consistent with a role of c-Abl in DSB repair regulation. In conjunction with these findings, transient abrogation of c-Abl activity resulted in increased cellular radioresistance. Our findings suggest a novel function for c-Abl in inhibition of the slow phase of DSB repair

Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

A Dynamic View of Domain-Motif Interactions

Many protein-protein interactions are mediated by domain-motif interaction, where a domain in one protein binds a short linear motif in its interacting partner. Such interactions are often involved in key cellular processes, necessitating their tight regulation. A common strategy of the cell to control protein function and interaction is by post-translational modifications of specific residues, especially phosphorylation. Indeed, there are motifs, such as SH2-binding motifs, in which motif phosphorylation is required for the domain-motif interaction. On the contrary, there are other examples where motif phosphorylation prevents the domain-motif interaction. Here we present a large-scale integrative analysis of experimental human data of domain-motif interactions and phosphorylation events, demonstrating an intriguing coupling between the two. We report such coupling for SH3, PDZ, SH2 and WW domains, where residue phosphorylation within or next to the motif is implied to be associated with switching on or off domain binding. For domains that require motif phosphorylation for binding, such as SH2 domains, we found coupled phosphorylation events other than the ones required for domain binding. Furthermore, we show that phosphorylation might function as a double switch, concurrently enabling interaction of the motif with one domain and disabling interaction with another domain. Evolutionary analysis shows that co-evolution of the motif and the proximal residues capable of phosphorylation predominates over other evolutionary scenarios, in which the motif appeared before the potentially phosphorylated residue, or vice versa. Our findings provide strengthening evidence for coupled interaction-regulation units, defined by a domain-binding motif and a phosphorylated residue

ABL fusion oncogene transformation and inhibitor sensitivity are mediated by the cellular regulator RIN1

ABL gene translocations create constitutively active tyrosine kinases that are causative in chronic myeloid leukemia, acute lymphocytic leukemia and other hematopoietic malignancies. Consistent retention of ABL SH3/SH2 autoinhibitory domains, however, suggests that these leukemogenic tyrosine kinase fusion proteins remain subject to regulation. We resolve this paradox, demonstrating that BCR-ABL1 kinase activity is regulated by RIN1, an ABL SH3/SH2 binding protein. BCR-ABL1 activity was increased by RIN1 overexpression and decreased by RIN1 silencing. Moreover, Rin1(−/−) bone marrow cells were not transformed by BCR-ABL1, ETV6-ABL1 or BCR-ABL1(T315I), a patient-derived drug-resistant mutant, as judged by growth factor independence. Rescue by ectopic RIN1 verified a cell autonomous mechanism of collaboration with BCR-ABL1 during transformation. Sensitivity to the ABL kinase inhibitor imatinib was increased by RIN1 silencing, consistent with RIN1 stabilization of an activated BCR-ABL1 conformation having reduced drug affinity. The dependence on activation by RIN1 to unleash full catalytic and cell transformation potential reveals a previously unknown vulnerability that could be exploited for treatment of leukemic cases driven by ABL translocations. The findings suggest that RIN1 targeting could be efficacious for imatinib-resistant disease and might complement ABL kinase inhibitors in first-line therapy

Sustained Modafinil Treatment Effects on Control-Related Gamma Oscillatory Power in Schizophrenia

Control-related cognitive processes such as rule selection and maintenance are associated with cortical oscillations in the gamma range, and modulated by catecholamine neurotransmission. Control-related gamma power is impaired in schizophrenia, and an understudied treatment target. It remains unknown whether pro-catecholamine pharmacological agents augment control-related gamma oscillations in schizophrenia. We tested the effects of 4-week fixed-dose daily adjunctive modafinil (MOD) 200 mg, in a randomized double-blind, placebo-controlled, parallel-groups design. Twenty-seven stable schizophrenia patients performed a cognitive control task during EEG, at baseline and after 4 weeks of treatment. EEG data underwent time-frequency decomposition with Morlet wavelets to determine power of 4–80 Hz oscillations. The modafinil group (n=14), relative to placebo group (n=13), exhibited enhanced oscillatory power associated with high-control rule selection in the gamma range after treatment, with additional effects during rule maintenance in gamma and sub-gamma ranges. MOD-treated patients who exhibited improved task performance with treatment also showed greater treatment-related delay period gamma compared with MOD-treated patients without improved performance. This is the first evidence in schizophrenia of augmentation of cognition-related gamma oscillations by an FDA-approved agent with therapeutic potential. Gamma oscillations represent a novel treatment target in this disorder, and modulation of catecholamine signaling may represent a viable strategy at this target

Modafinil Effects on Middle-Frequency Oscillatory Power During Rule Selection in Schizophrenia

Control-related cognitive processes such as rule selection are associated with cortical oscillations in the theta, alpha and, beta ranges, and modulated by catecholamine neurotransmission. Thus, a potential strategy for improving cognitive control deficits in schizophrenia would be to use pro-catecholamine pharmacological agents to augment these control-related oscillations. In a double-blind, placebo-controlled (within-subjects) study, we tested the effects of adjunctive single-dose modafinil 200 mg on rule-related 4–30 Hz oscillations in 23 stable schizophrenia patients, using EEG during cognitive control task performance. EEG data underwent time-frequency decomposition with Morlet wavelets to determine the power of 4–30 Hz oscillations. Modafinil (relative to placebo) enhanced oscillatory power associated with high-control rule selection in theta, alpha, and beta ranges, with modest effects during rule maintenance. Modafinil treatment in schizophrenia augments middle-frequency cortical oscillatory power associated with rule selection, and may subserve diverse subcomponent processes in proactive cognitive control