Yasuko Tomono

IDENTIFICATION OF A NOVEL PHOSPHORYLATION SITE ON HISTONE H3 COUPLED WITH MITOTIC CHROMOSOME CONDENSATION

Histone H3 (H3) phosphorylation at Ser10 occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation inTetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr7, we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser28phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser28. This antibody specifically recognized the phosphorylation of H3 Ser28 but not that of glial fibrillary acidic protein Thr7. Immunocytochemical studies with HTA28 revealed that Ser28 phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using32P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser28 during early mitosis. In addition, we found that H3 is phosphorylated at Ser28 as well as Ser10 when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser28, together with Ser10, is a conserved event and is likely to be involved in mitotic chromosome condensation.

Anti-high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats

The high mobility group box‐1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine‐like activity in the extracellular space. Here we show that treatment with neutralizing anti‐HMGB1 monoclonal antibody (mAb; 200 μg, twice) remarkably ameliorated brain infarction induced by 2‐h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti‐HMGB1 mAb inhibited the increased permeability of the blood‐brain barrier, the activation of microglia, the expression of TNF‐α and iNOS, and suppressed the activity of MMP‐9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemi‐cal studyrevealed that HMGB1 immunoreactivityin the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an out‐standingly suitable target for the treatment. Intravenous injection of neutralizing anti‐HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.— Liu, K., Mori, S., Takahashi, H. K., Tomono, Y., Wake, H., Kanke, T., Sato, Y., Hiraga, N., Adachi, N., Yoshino, T., Nishibori, M. Anti‐high mobility group box 1 monoclonal antibody ameliorates brain infarction induced by transient ischemia in rats. FASEB J. 21, 3904–3916 (2007)

Complex formation of Plk1 and INCENP required for metaphase-anaphase transition

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis : cleavage furrow-specific phosphorylation of intermediate filaments

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

ERBIN associates with p0071, an armadillo protein, at cell‐cell junctions of epithelial cells

Working model for the ERBIN–p0071 interaction at cell‐cell adhesions. ERBIN is associated with p0071 at adherens junctions and desmosomes. p0071 is thought to interact with classic cadherins or desmosomal cadherins ( Hatzfeld 1999 ). The subcellular localization of ERBIN may be regulated by the Rho family and other signals controlling cell polarity.

Inhibition of focal adhesion kinase as a potential therapeutic strategy for imatinib-resistant gastrointestinal stromal tumor

Focal adhesion kinase (FAK) is often up-regulated in a variety of malignancies, including gastrointestinal stromal tumor (GIST), and its overexpression seems to be associated with tumor progressiveness and poor prognosis. GIST is well known to have a mutation to c-KIT; thus, a specific c-KIT inhibitor (imatinib) is recognized as the first-line chemotherapy for GIST, although a certain type of c-KIT mutation reveals a resistance to imatinib due to as yet uncertain molecular mechanisms. To assess the c-KIT mutation-related variation of cellular responses to imatinib, murine lymphocyte-derived Ba/F3 cells, which are stably transduced with different types of c-KIT mutation, were treated with either imatinib or a FAK inhibitor (TAE226), and their antitumor effects were determined in vitro and in vivo. A mutation at exon 11 (KITdel559-560) displayed a high sensitivity to imatinib, whereas that at exon 17 (KIT820Tyr) showed a significant resistance to imatinib in vitro and in vivo. KIT820Tyr cells appeared to maintain the activities of FAK and AKT under the imatinib treatment, suggesting that FAK might play a role in cell survival in imatinib-resistant cells. When FAK activity in those cells was inhibited by TAE226, cell growth was equally suppressed and the cells underwent apoptosis regardless of the c-KIT mutation types. Oral administration of TAE226 significantly diminished tumor growth in nude mice bearing KIT820Tyr xenografts. In summary, c-KIT mutation at exon 17 displayed a resistance to imatinib with maintained activations of FAK and subsequent survival signals. Targeting FAK could be a potential therapeutic strategy for imatinib-resistant GISTs. [Mol Cancer Ther 2009;8(1):127–34]

14-3-3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.

Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G2/M transition. We reported recently that Chk1 is phosphorylated at Ser286 and Ser301 by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser286 and Ser301 to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B1-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B1 and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.

Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1)

Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase‐anaphase transition. Two‐dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin‐dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1‐S301 phosphorylation, although treatment with p38‐specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole‐arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.

Dual-Tyrosine Kinase Inhibitor for Focal Adhesion Kinase and Insulin-like Growth Factor-I Receptor Exhibits Anticancer Effect in Esophageal Adenocarcinoma In vitro and In vivo

Purpose: Focal adhesion kinase (FAK) regulates integrin and growth factor–mediated signaling pathways to enhance cell migration, proliferation, and survival, and its up-regulation correlates malignant grade and poor outcome in several types of cancer. In this study, we aimed to raise a potential therapeutic strategy using a FAK inhibitor for Barretts esophageal adenocarcinoma. Experimental Design: The expression status of FAK in clinical Barretts esophageal adenocarcinoma tissues was determined by immunohistochemistry. Cultured esophageal adenocarcinoma cells were treated with TAE226, a specific FAK inhibitor with an additional effect of inhibiting insulin-like growth factor-I receptor (IGF-IR), to assess its anticancer effect in vitro. Western blot was carried out to explore a participating signaling pathway for TAE226-induced cell death. Furthermore, TAE226 was orally administered to s.c. xenograft animals to investigate its anticancer effect in vivo. Results: Strong expression of FAK was found in 94.0% of Barretts esophageal adenocarcinoma compared with 17.9% of Barretts epithelia, suggesting that FAK might play a critical role in the progression of Barretts esophageal adenocarcinoma. When esophageal adenocarcinoma cells were treated with TAE226, cell proliferation and migration were greatly inhibited with an apparent structural change of actin fiber and a loss of cell adhesion. The activities of FAK, IGF-IR, and AKT were suppressed by TAE226 and subsequent dephosphorylation of BAD at Ser136 occurred, resulting in caspase-mediated apoptosis. In vivo tumor volume was significantly reduced by oral administration of TAE226. Conclusions: These results suggest that TAE226, a dual tyrosine kinase inhibitor for FAK and IGF-IR, could become a new remedy for Barretts esophageal adenocarcinoma.