Ryuzaburo Yuki

Phosphorylation of KRAB-associated Protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by Nuclear Tyrosine Kinases Inhibits the Association of KAP1 and Heterochromatin Protein 1α (HP1α) with Heterochromatin

Background: We showed that nuclear tyrosine phosphorylation is involved in chromatin structural changes. Results: Several tyrosine kinases phosphorylate KAP1 at Tyr-449, Tyr-458, and Tyr-517 in the nucleus, resulting in a decrease of KAP1 association with heterochromatin. Conclusion: Tyrosine phosphorylation of KAP1 by nucleus-localized tyrosine kinases, including Src, involves heterochromatin structural changes. Significance: These findings provide a new insight into nuclear tyrosine phosphorylation signals. Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1–3YF). In DNA damage, KAP1–3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.

Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation.

c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Background: Mimosine is a cell synchronization reagent used for arresting cells in late G1 and S phases. Results: Replication fork assembly is reversibly blocked by ATM activation through mimosine-generated reactive oxygen species. Conclusion: Mimosine induces cell cycle arrest strictly at the G1-S phase boundary, which prevents replication fork stalling-induced DNA damage. Significance: These findings provide a novel mechanism of the mimosine-induced G1 checkpoint. Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

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.

Cdk1-mediated phosphorylation of human ATF7 at Thr-51 and Thr-53 promotes cell-cycle progression into M phase.

Activating transcription factor 2 (ATF2) and its homolog ATF7 are phosphorylated at Thr-69/Thr-71 and at Thr-51/Thr-53, respectively, by stress-activated MAPKs regulating their transcriptional functions in G1 and S phases. However, little is known about the role of ATF2 and ATF7 in G2/M phase. Here, we show that Cdk1-cyclin B1 phosphorylates ATF2 at Thr-69/Thr-71 and ATF7 at Thr-51/Thr-53 from early prophase to anaphase in the absence of any stress stimulation. Knockdown of ATF2 or ATF7 decreases the rate of cell proliferation and the number of cells in M-phase. In particular, the knockdown of ATF7 severely inhibits cell proliferation and G2/M progression. The inducible expression of a mitotically nonphosphorylatable version of ATF7 inhibits G2/M progression despite the presence of endogenous ATF7. We also show that mitotic phosphorylation of ATF7 promotes the activation of Aurora kinases, which are key enzymes for early mitotic events. These results suggest that the Cdk1-mediated phosphorylation of ATF7 facilitates G2/M progression, at least in part, by enabling Aurora signaling.

Fyn Accelerates M Phase Progression by Promoting the Assembly of Mitotic Spindle Microtubules

The mitotic spindle is the major piece of cellular machinery essential for faithful chromosome segregation. Whereas Fyn, a member of Src‐family kinases, is known to be localized to the meiotic and mitotic spindle microtubules, the role of Fyn in mitotic spindle formation has not yet been completely elucidated. In this study, we studied the role of Fyn in spindle formation and effects on M‐phase progression. Re‐expression of Fyn induced increases in the fluorescence intensity of mitotic spindle microtubules in SYF cells having triple knock‐out mutations of c‐Src, c‐Yes, and Fyn. Cold treatment results showed that Fyn increases the maximum length of microtubules in HeLa S3 cells in a manner dependent on Fyn kinase activity. Complete depolymerization of microtubules under cold treatment and the following release into 37°C revealed that the increase in the microtubule length in Fyn‐expressing cells may be attributed to the promotion of microtubule polymerization. After cold treatment, Fyn promotes the accumulation of EB1, which is a plus‐end tracking protein and facilitates microtubule growth, in a manner dependent on the kinase activity. Furthermore, Fyn accelerates the M phase progression of cells from nocodazole arrest. These results suggest that Fyn facilitates mitotic spindle formation through the increase in microtubule polymerization, resulting in the acceleration of M‐phase progression. J. Cell. Biochem. 117: 894–903, 2016.

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.

Protective role for lipid modifications of Src-family kinases against chromosome missegregation

Src-family tyrosine kinases, which are expressed in various cell types, play critical roles in cell signalling at the cytoplasmic side of the plasma membrane through their lipid modifications. Src-family kinases are cotranslationally myristoylated and posttranslationally palmitoylated in the amino-terminal region. The Src-family member Lyn contains a myristoylation site at glycine-2 and a palmitoylation site at cysteine-3, whereas c-Src has a myristoylation site at glycine-2 but not any palmitoylation sites. However, little is known about the role for lipid modifications of Src-family kinases in cell division. Here, we show that non-lipid-modified Lyn and c-Src, Lyn(G2A/C3A) and c-Src(G2A), are delocalized from membranes to the cytoplasm and the nucleus, which gives rise to a significant increase in the rate of chromosome missegregation, such as chromosome lagging and anaphase chromosome bridging, in a tyrosine kinase activity-dependent manner. Treatment with the Src inhibitor PP2 shows that the kinase activity of non-lipid-modified, non-membrane-bound Src during M phase is critical for giving rise to chromosome missegregation. Given that only a fraction of Src-family kinases fails in lipid modifications during biosynthesis, these results suggest that Src’s membrane anchorage through their lipid modifications from prophase to anaphase plays a protective role against induction of chromosome missegregation.

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.