Phosphotyrosine proteomics in cells synchronized at monopolar cytokinesis reveals EphA2 as functioning in cytokinesis.

Cytokinesis is the final step of the cell division in which cellular components are separated into two daughter cells. This process is regulated through the phosphorylation of different classes of proteins by serine/threonine (Ser/Thr) kinases such as Aurora B and Polo-like kinase 1 (PLK1). Conversely, the role of phosphorylation at tyrosine residues during cytokinesis has not been studied in detail yet. In this study, we performed a phosphotyrosine proteomic analysis of cells undergoing monopolar cytokinesis synchronized by using the Eg5 inhibitor (+)-S-trityl-l-cysteine (STLC) and the CDK1 inhibitor RO-3306. Phosphotyrosine proteomics gave 362 tyrosine-phosphorylated peptides. Western blot analysis of proteins revealed tyrosine phosphorylation in mitogen-activated protein kinase 14 (MAPK14), vimentin, ephrin type-A receptor 2 (EphA2), and myelin protein zero-like protein 1 (MPZL1) during monopolar cytokinesis. Additionally, we demonstrated that EphA2, a protein with unknown function during cytokinesis, is involved in cytokinesis. EphA2 knockdown accelerated epithelial cell transforming 2 (Ect2) knockdown-induced multinucleation, suggesting that EphA2 plays a role in cytokinesis in a particular situation. The list also included many proteins previously reported to play roles during cytokinesis. These results evidence that the identified phosphopeptides facilitate the identification of novel tyrosine phosphorylation signaling involved in regulating cytokinesis.

The conserved C-terminal residues of FAM83H are required for the recruitment of casein kinase 1 to the keratin cytoskeleton.

The casein kinase 1 (CK1) family of serine/threonine protein kinases is involved in diverse cellular events at discrete subcellular compartments. FAM83H acts as a scaffold protein that recruits CK1 to the keratin cytoskeleton or to the nuclear speckles, which are storage sites for splicing factors. We determined the amino acid region of FAM83H required for recruiting CK1 to the keratin cytoskeleton. The subcellular localization of mutant FAM83H proteins with deletions of amino acid residues at different positions was evaluated via immunofluorescence. FAM83H mutants with deleted C-terminal residues 1134-1139, which are conserved among vertebrates, lost the ability to localize and recruit CK1 to the keratin cytoskeleton, suggesting that these residues are required for recruiting CK1 to the keratin cytoskeleton. The deletion of these residues (1134-1139) translocated FAM83H and CK1 to the nuclear speckles. Amino acid residues 1 to 603 of FAM83H were determined to contain the region responsible for the recruitment of CK1 to the nuclear speckles. Our results indicated that FAM83H recruits CK1 preferentially to the keratin cytoskeleton and alternatively to the nuclear speckles.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

v-Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v-Src-mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities-mediated chromosome instability. v-Src directly phosphorylates Tyr-15 of cyclin-dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v-Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v-Src restores cancer cell viability reduced by various microtubule-targeting agents (MTAs), although v-Src does not alter cytotoxicity of DNA-damaging anticancer drugs. v-Src causes mitotic slippage of MTAs-treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v-Src also restores cell viability reduced by a polo-like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v-Src. These results suggest that the v-Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src-induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Src-mediated tyrosine phosphorylation of PRC1 and kinastrin/SKAP on the mitotic spindle.

Src-family tyrosine kinases (SFKs) play important roles in a number of signal transduction events during mitosis, such as spindle formation. A relationship has been reported between SFKs and the mitotic spindle; however, the underlying mechanisms remain unclear. We herein demonstrated that SFKs accumulated in the centrosome region at the onset of mitosis. Centrosomal Fyn increased in the G2 phase in a microtubule polymerization-dependent manner. A mass spectrometry analysis using mitotic spindle preparations was performed to identify tyrosine-phosphorylated substrates. Protein regulator of cytokinesis 1 (PRC1) and kinastrin/small kinetochore-associated protein (kinastrin/SKAP) were identified as SFK substrates. SFKs mainly phosphorylated PRC1 at Tyr-464 and kinastrin at Tyr-87. Although wild-type PRC1 is associated with microtubules, phosphomimetic PRC1 impaired the ability to bind microtubules. Phosphomimetic kinastrin at Tyr-87 also impaired binding with microtubules. Collectively, these results suggest that tyrosine phosphorylation of PRC1 and kinastrin plays a role in their delocalization from microtubules during mitosis.

Binding of Cu2+ to Aß1-29 causes aggregation and toxicity in SH-SY5Y cells.

The accumulation and aggregation of amyloid-ß (Aß) are critical factors in the pathogenesis of Alzheimer's disease (AD). Several studies have indicated that metal ions such as Cu2+and Zn2+ play a key role in the formation and stabilization of neurotoxic Aß aggregates, however the molecular mechanisms underlying Aß cytotoxicity have not yet been fully elucidated. Previously, we showed that the Aß-derived fragment peptide (Aß-FrP), Aß1-19, altered conformation in the presence of Cu2+, inhibiting its digestion by metalloproteinase-7 (MMP-7). In this study we demonstrated that Aß1-19 did not form aggregates in the presence of Cu2+. Therefore, we synthesized a new Aß-FrP, Aß1-29, which displayed Cu2+-dependent conformational conversion and aggregate formation. Aß1-29 was cleaved by MMP-7, however this reaction was inhibited in the presence of Cu2+ in a similar way to Aß1-19. Interestingly, Aß1-29 showed conformational conversion and aggregate formation in the presence of Zn2+, however this did not confer resistance against MMP-7 cleavage. Moreover, Aß1-29 induced the apoptotic cell death of neural SH-SY5Y cells in the presence of Cu2+ but not Zn2+. These results suggest that Cu2+, unlike Zn2+, may play an important role in the aggregation mechanism of Aß and thus in the pathology of AD.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Depletion of Csk preferentially reduces the protein level of LynA in a Cbl-dependent manner in cancer cells.

There are eight human Src-family tyrosine kinases (SFKs). SFK members c-Src, c-Yes, Fyn, and Lyn are expressed in various cancer cells. SFK kinase activity is negatively regulated by Csk tyrosine kinase. Reduced activity of Csk causes aberrant activation of SFKs, which can be degraded by a compensatory mechanism depending on Cbl-family ubiquitin ligases. We herein investigated whether all SFK members are similarly downregulated by Cbl-family ubiquitin ligases in cancer cells lacking Csk activity. We performed Western blotting of multiple cancer cells knocked down for Csk and found that the protein levels of the 56 kDa isoform of Lyn (LynA), 53 kDa isoform of Lyn (LynB), c-Src, and Fyn, but not of c-Yes, were reduced by Csk depletion. Induction of c-Cbl protein levels was also observed in Csk-depleted cells. The reduction of LynA accompanying the depletion of Csk was significantly reversed by the knockdown for Cbls, whereas such significant recovery of LynB, c-Src, and Fyn was not observed. These results suggested that LynA is selectively downregulated by Cbls in cancer cells lacking Csk activity.

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation.

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105ß exhibit distinct functions with different subcellular localizations. Hsp105ß localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105ß is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105ß is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.

Desuppression of TGF-ß signaling via nuclear c-Abl-mediated phosphorylation of TIF1γ/TRIM33 at Tyr-524, -610, and -1048.

Protein-tyrosine kinases regulate a broad range of intracellular processes occurring primarily just beneath the plasma membrane. With the greatest care to prevent dephosphorylation, we have shown that nuclear tyrosine phosphorylation regulates global chromatin structural states. However, the roles for tyrosine phosphorylation in the nucleus are poorly understood. Here we identify transcriptional intermediary factor 1-γ (TIF1γ/TRIM33/Ectodermin), which suppresses transforming growth factor-ß (TGF-ß) signaling through the association with Smad2/3 transcription factor, as a new nuclear substrate of c-Abl tyrosine kinase. Replacement of the three tyrosine residues Tyr-524, -610, and -1048 with phenylalanine (3YF) inhibits c-Abl-mediated phosphorylation of TIF1γ and enhances TIF1γ's association with Smad3. Importantly, knockdown-rescue experiments show that 3YF strengthens TIF1γ's ability to suppress TGF-ß signaling. Intriguingly, activation of c-Abl by epidermal growth factor (EGF) induces desuppression of TGF-ß signaling via enhancing the tyrosine phosphorylation level of TIF1γ. TGF-ß together with EGF synergistically provokes desuppressive responses of epithelial-to-mesenchymal transition through tyrosine phosphorylation of TIF1γ. These results suggest that nuclear c-Abl-mediated tyrosine phosphorylation of TIF1γ has a desuppressive role in TGF-ß-Smad2/3 signaling.

The tyrosine kinase v-Src causes mitotic slippage by phosphorylating an inhibitory tyrosine residue of Cdk1.

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and in vitro kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.

Air-drying of cells enables visualization of antiparallel microtubule overlaps in the spindle midzone.

Immunofluorescence staining is used extensively to examine various types of cellular events. However, even when an antibody can detect its epitopes in western blotting, it sometimes fails to detect its epitopes when used for immunofluorescence staining. One example is the antiparallel microtubule overlaps in the anaphase and telophase spindle midzone, which functions as a signaling scaffold for cleavage furrow specification. It has been believed that it cannot be visualized by immunofluorescence staining due to the highly dense structure of microtubule overlaps (Ifuji et al., 2017). Here, we show a simple method for visualization of antiparallel microtubule overlaps in the anaphase and telophase spindle midzone. •Air-drying cells before fixation enables visualization of antiparallel microtubule overlaps in the anaphase and telophase spindle midzone, which cannot be visualized by the conventional method.•Simple method that requires minimal usage of equipment.•Commonly used anti-tubulin antibodies can be used in this method.

Deep Phospho- and Phosphotyrosine Proteomics Identified Active Kinases and Phosphorylation Networks in Colorectal Cancer Cell Lines Resistant to Cetuximab.

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.

A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone.

Cell division, in which duplicated chromosomes are separated into two daughter cells, is the most dynamic event during cell proliferation. Chromosome movement is powered mainly by microtubules, which vary in morphology and are organized into characteristic structures according to mitotic progression. During the later stages of mitosis, antiparallel microtubules form the spindle midzone, and the irregular formation of the midzone often leads to failure of cytokinesis, giving rise to the unequal segregation of chromosomes. However, it is difficult to analyze the morphology of these microtubules because microtubules in the antiparallel overlaps of microtubule-plus ends in the midzone are embedded in highly electron-dense matrices, impeding the access of anti-tubulin antibodies to their epitopes during immunofluorescence staining. Here, we developed a novel method to visualize selectively antiparallel microtubule overlaps in the midzone. When cells are air-dried before fixation, aligned α-tubulin staining is observed and colocalized with PRC1 in the center of the midzone of anaphase and telophase cells, suggesting that antiparallel microtubule overlaps can be visualized by this method. In air-dried cells, mCherry-α-tubulin fluorescence and ß-tubulin staining show almost the same pattern as α-tubulin staining in the midzone, suggesting that the selective visualization of antiparallel microtubule overlaps in air-dried cells is not attributed to an alteration of the antigenicity of α-tubulin. Taxol treatment extends the microtubule filaments of the midzone in air-dried cells, and nocodazole treatment conversely decreases the number of microtubules, suggesting that unstable microtubules are depolymerized during the air-drying method. It is of note that the air-drying method enables the detection of the disruption of the midzone and premature midzone formation upon Aurora B and Plk1 inhibition, respectively. These results suggest that the air-drying method is suitable for visualizing microtubules in the antiparallel overlaps of microtubule-plus ends of the midzone and for detecting their effects on midzone formation.

Enhancement of TGF-ß-induced Smad3 activity by c-Abl-mediated tyrosine phosphorylation of its coactivator SKI-interacting protein (SKIP).

c-Abl is a non-receptor-type tyrosine kinase that plays an important role in cell proliferation, migration, apoptosis, and fibrosis. Furthermore, although c-Abl is involved in transforming growth factor-ß (TGF-ß) signaling, its molecular functions in TGF-ß signaling are not fully understood. Here, we found that c-Abl phosphorylates SKI-interacting protein (SKIP), a nuclear cofactor of the transcription factor Smad3. The c-Abl inhibitor imatinib suppressed TGF-ß-induced expression of Smad3 targets as well as SKIP/Smad3 interaction. TGF-ß-stimulation induced tyrosine phosphorylation of SKIP, and this phosphorylation was suppressed by imatinib. Tyr292, Tyr430, and Tyr433 residues in SKIP were shown to be involved in c-Abl-mediated phosphorylation. Phosphomimetic glutamic acid substitution at Tyr292 in SKIP enhanced, whereas its phospho-dead phenylalanine substitution attenuated TGF-ß-induced SKIP/Smad3 interaction. Moreover, the phosphomimetic mutant of SKIP augmented transcriptional activity of Smad3. Taken together, these results suggest that c-Abl phosphorylates SKIP mainly at Tyr292 and promotes SKIP/Smad3 interaction for the full activation of TGF-ß/Smad3 signaling.

Requirement of Hsp105 in CoCl2-induced HIF-1α accumulation and transcriptional activation.

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl2. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl2 induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl2 treatment. The CoCl2-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl2-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl2-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α.

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.

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. © 2016 Wiley Periodicals, Inc.

v-Src-induced nuclear localization of YAP is involved in multipolar spindle formation in tetraploid cells.

The protein-tyrosine kinase, c-Src, is involved in a variety of signaling events, including cell division. We have reported that v-Src, which is a mutant variant of the cellular proto-oncogene, c-Src, causes delocalization of Aurora B kinase, resulting in a furrow regression in cytokinesis and the generation of multinucleated cells. However, the effect of v-Src on mitotic spindle formation is unknown. Here we show that v-Src-expressing HCT116 and NIH3T3 cells undergo abnormal cell division, in which cells separate into more than two cells. Upon v-Src expression, the proportion of multinucleated cells is increased in a time-dependent manner. Flow cytometry analysis revealed that v-Src increases the number of cells having a ≥4N DNA content. Microscopic analysis showed that v-Src induces the formation of multipolar spindles with excess centrosomes. These results suggest that v-Src induces multipolar spindle formation by generating multinucleated cells. Tetraploidy activates the tetraploidy checkpoint, leading to a cell cycle arrest of tetraploid cells at the G1 phase, in which the nuclear exclusion of the transcription co-activator YAP plays a critical role. In multinucleated cells that are induced by cytochalasin B and the Plk1 inhibitor, YAP is excluded from the nucleus. However, v-Src prevents this nuclear exclusion of YAP through a decrease in the phosphorylation of YAP at Ser127 in multinucleated cells. Furthermore, v-Src decreases the expression level of p53, which also plays a critical role in the cell cycle arrest of tetraploid cells. These results suggest that v-Src promotes abnormal spindle formation in at least two ways: generation of multinucleated cells and a weakening of the tetraploidy checkpoint.