Mariko Morii

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

Background: Once DNA repair is completed, the DNA damage checkpoint is terminated, and the cell cycle is resumed. Results: Src inhibition induced a delay in G2 checkpoint recovery and persistent ATR-Chk1 activation. Conclusion: Src inhibits ATR signaling to promote recovery from G2 checkpoint arrest. Significance: Src sends a termination signal between the completion of DNA repair and the initiation of checkpoint termination. The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination.

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Background: Tyrosine kinases are active in the cell nucleus and involved in global nuclear structure. Results: Phosphorylation of AKAP8 at multiple tyrosine residues by several nucleus-localized tyrosine kinases, including c-Src, induces AKAP8s dissociation from nuclear structures. Conclusion: Nuclear tyrosine phosphorylation of AKAP8 is involved in global nuclear structure changes. Significance: These findings highlight the importance of nuclear tyrosine phosphorylation in dynamic chromatin regulation. Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.

c-Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity-dependent manner.

c‐Abl is a non‐receptor‐type tyrosine kinase that regulates various cellular events, including cell proliferation, differentiation, and apoptosis, through phosphorylation of cytoplasmic and nuclear targets. Although we showed that c‐Abl induces histone deacetylation, the molecular mechanisms of this phenomenon are largely unknown. Here, we analyzed the effect of c‐Abl on the expression of histone deacetylase 1 (HDAC1), because c‐Abl was shown to be involved in maintenance of nuclear protein levels of HDAC1. Co‐transfection of HDAC1 with c‐Abl increased the levels of HDAC1 protein in a kinase activity‐dependent manner without affecting its mRNA levels. Treatment with the proteasome inhibitor MG132 increased protein levels of HDAC1 in cells transfected with HDAC1 but not in cells co‐transfected with HDAC1 and c‐Abl. Among class I HDACs, knockdown of endogenous c‐Abl preferentially suppressed endogenous protein levels of HDAC1, suggesting that c‐Abl stabilizes HDAC1 protein by inhibiting its proteasomal degradation. Subcellular fractionation showed that the stabilization of HDAC1 by c‐Abl occurred in the nucleus. Despite the fact that HDAC1 was phosphorylated by co‐expression with c‐Abl, stabilization of HDAC1 by c‐Abl was not affected by mutations in its sites phosphorylated by c‐Abl. Co‐expression with HDAC1 and nuclear‐targeted c‐Abl did not affect HDAC1 stabilization. Therefore, these results suggest that c‐Abl induces HDAC1 stabilization possibly through phosphorylation of a cytoplasmic target that is involved in proteasomal degradation of HDAC1.

Imatinib inhibits inactivation of the ATM/ATR signaling pathway and recovery from adriamycin/doxorubicin-induced DNA damage checkpoint arrest

The DNA damage checkpoint arrests cell cycle progression to allow time for DNA repair. After completion of DNA repair, checkpoint activation is terminated, and cell cycle progression is resumed in a process called checkpoint recovery. The activation of the checkpoint has been studied in depth, but little is known about recovery from the DNA damage checkpoint. Recently we showed that Src family kinases promote recovery from the G2 DNA damage checkpoint. Here we show that imatinib inhibits inactivation of ATM/ATR signaling pathway to suppress recovery from Adriamycin/doxorubicin‐induced DNA damage checkpoint arrest. Imatinib and pazopanib, two distinct inhibitors of PDGFR/c‐Kit family kinases, delayed recovery from checkpoint arrest and inhibited the subsequent S–G2–M transition after Adriamycin exposure. By contrast, imatinib and pazopanib did not delay the recovery from checkpoint arrest in the presence of an ATM/ATR inhibitor caffeine. Consistently, imatinib induced a persistent activation of ATR–Chk1 signaling. By the way, the maintenance of G2 checkpoint arrest is largely dependent on ATR–Chk1 signaling. However, unlike Src inhibition, imatinib did not delay the recovery from checkpoint arrest in the presence of an ATM inhibitor KU‐55933. Furthermore, imatinib induced a persistent activation of ATM–KAP1 signaling, and a possible involvement of imatinib in an ATM‐dependent DNA damage response is suggested. These results reveal that imatinib inhibits recovery from Adriamycin‐induced DNA damage checkpoint arrest in an ATM/ATR‐dependent manner and raise the possibility that imatinib may inhibit resumption of tumor proliferation after chemo‐ and radiotherapy.

v-Src inhibits the interaction between Rad17 and Rad9 and induces replication fork collapse.

ATR-dependent DNA damage checkpoint is crucial to maintain genomic stability. Recently, we showed that Src family kinases suppress ATR-dependent checkpoint signaling in termination of DNA damage checkpoint. However, the precise molecular mechanism is unclear. Therefore, we examined the role of oncogenic v-Src on ATR-Chk1 signaling. We show that v-Src suppresses thymidine-induced Chk1 phosphorylation and induces replication fork collapse. v-Src inhibits interaction between Rad17 and Rad9 in chromatin fraction. By contrast, v-Src does not inhibit RPA32 phosphorylation, ATR autophosphorylation, or TopBP1-Rad9 interaction. These data suggest that v-Src attenuates ATR-Chk1 signaling through the inhibition of Rad17-Rad9 interaction.

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non‐receptor‐type tyrosine kinase c‐Abl. c‐Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N‐ and C‐terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C‐terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c‐Abl, enhanced the activation of estrogen signaling. In contrast, the c‐Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild‐type. These results suggest that c‐Abl‐mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453–1461, 2017.

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

Src Acts as an Effector for Ku70-dependent Suppression of Apoptosis through Phosphorylation of Ku70 at Tyr-530

Src-family tyrosine kinases are widely expressed in many cell types and participate in a variety of signal transduction pathways. Despite the significance of Src in suppression of apoptosis, its mechanism remains poorly understood. Here we show that Src acts as an effector for Ku70-dependent suppression of apoptosis. Inhibition of endogenous Src activity promotes UV-induced apoptosis, which is impaired by Ku70 knockdown. Src phosphorylates Ku70 at Tyr-530, being close to the possible acetylation sites involved in promotion of apoptosis. Src-mediated phosphorylation of Ku70 at Tyr-530 decreases acetylation of Ku70, whereas Src inhibition augments acetylation of Ku70. Importantly, knockdown-rescue experiments with stable Ku70 knockdown cells show that the nonphosphorylatable Y530F mutant of Ku70 reduces the ability of Ku70 to suppress apoptosis accompanied by augmentation of Ku70 acetylation. Our results reveal that Src plays a protective role against hyperactive apoptotic cell death by reducing apoptotic susceptibility through phosphorylation of Ku70 at Tyr-530.

Overexpression of zinc-finger protein 777 (ZNF777) inhibits proliferation at low cell density through down-regulation of FAM129A.

Krüppel‐associated box‐containing zinc finger proteins (KRAB‐ZFPs) regulate a wide range of cellular processes. KRAB‐ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB‐ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777‐mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777‐mediated down‐regulation of FAM129A induces moderate levels of the cyclin‐dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777‐mediated down‐regulation of FAM129A plays a role in inhibition of cell proliferation. J. Cell. Biochem. 116: 954–968, 2015.