Kiyotsugu Yoshida

Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage

BRCA1 (BReast‐CAncer susceptibility gene 1) and BRCA2 are tumor suppressor genes, the mutant phenotypes of which predispose to breast and ovarian cancers. Intensive research has shown that BRCA proteins are involved in a multitude of pivotal cellular processes. In particular, both genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Recent studies suggest that BRCA proteins are required for maintenance of chromosomal stability, thereby protecting the genome from damage. New data also show that BRCAs transcriptionally regulate some genes involved in DNA repair, the cell cycle, and apo ptosis. Many of these functions are mediated by a large number of cellular proteins that interact with BRCAs. The functions of BRCA proteins are also linked to distinct and specific phosphory‐lation events; however, the extent to which phosphorylation‐acti‐vated molecular pathways contribute to tumor suppressor activity remains unclear. Finally, the reasons why mutations in BRCA genes lead to the development of breast and ovarian cancers are not clearly understood. Elucidation of the precise molecular functions of BRCAs is expected to improve our understanding of hereditary as well as sporadic mammary carcinogenesis.

JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage

The ubiquitously expressed c-Abl tyrosine kinase localizes to the cytoplasm and nucleus. Nuclear c-Abl is activated by diverse genotoxic agents and induces apoptosis; however, the mechanisms that are responsible for nuclear targeting of c-Abl remain unclear. Here, we show that cytoplasmic c-Abl is targeted to the nucleus in the DNA damage response. The results show that c-Abl is sequestered into the cytoplasm by binding to 14-3-3 proteins. Phosphorylation of c-Abl on Thr 735 functions as a site for direct binding to 14-3-3 proteins. We also show that, in response to DNA damage, activation of the c-Jun N-terminal kinase (Jnk) induces phosphorylation of 14-3-3 proteins and their release from c-Abl. Together with these results, expression of an unphosphorylated 14-3-3 mutant attenuates DNA-damage-induced nuclear import of c-Abl and apoptosis. These findings indicate that 14-3-3 proteins are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress.

Protein Kinase C δ Regulates Ser46 Phosphorylation of p53 Tumor Suppressor in the Apoptotic Response to DNA Damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser46 was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser46. Here, we show that the pro-apoptotic kinase, protein kinase C δ (PKCδ), is involved in phosphorylation of p53 on Ser46. PKCδ-mediated phosphorylation is required for the interaction of PKCδ with p53. The results also demonstrate that p53DINP1 associates with PKCδ upon exposure to genotoxic agents. Consistent with these results, PKCδ potentiates p53-dependent apoptosis by Ser46 phosphorylation in response to genotoxic stress. These findings indicate that PKCδ regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage.

ABSTRACT The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-xL protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.

Protein Kinase C δ Induces Transcription of the TP53 Tumor Suppressor Gene by Controlling Death-Promoting Factor Btf in the Apoptotic Response to DNA Damage

ABSTRACT Expression of the TP53 tumor suppressor is tightly controlled for its ability to function as a critical regulator of cell growth, proliferation, and death in response to DNA damage. However, little is known about the mechanisms and contributions of the transcriptional regulation of TP53. Here we report that protein kinase C δ (PKCδ), a ubiquitously expressed member of the novel subfamily of PKC isoforms, transactivates TP53 expression at the transcriptional level. Reporter assays demonstrated that PKCδ induces the promoter activity of TP53 through the TP53 core promoter element (CPE-TP53) and that such induction is enhanced in response to DNA damage. The results also demonstrate that, upon exposure to genotoxic stress, PKCδ activates and interacts with the death-promoting transcription factor Btf to co-occupy CPE-TP53. Inhibition of PKCδ activity decreases the affinity of Btf for CPE-TP53, thereby reducing TP53 expression at both the mRNA and the protein levels. In concert with these results, we show that disruption of Btf-mediated TP53 gene transcription by RNA interference leads to suppression of TP53-mediated apoptosis following genotoxic stress. These findings provide evidence that activation of TP53 gene transcription by PKCδ triggers TP53-dependent apoptosis in response to DNA damage.

Activation of SAPK/JNK Signaling by Protein Kinase Cδ in Response to DNA Damage

The cellular response to genotoxic stress includes activation of protein kinase Cδ (PKCδ). The functional role of PKCδ in the DNA damage response is unknown. The present studies demonstrate that PKCδ is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1-β-d-arabinofuranosylcytosine (araC) and other genotoxic agents. DNA damage-induced SAPK activation was attenuated by (i) treatment with rottlerin, (ii) expression of a kinase-inactive PKCδ(K-R) mutant, and (iii) down-regulation of PKCδ by small interfering RNA (siRNA). Coexpression studies demonstrate that PKCδ activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1 → MKK7 → SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKCδ, and in turn, MEKK1 is activated by a PKCδ-dependent mechanism. These findings indicate that PKCδ functions in the activation of SAPK through a Lyn → PKCδ → MEKK1 → MKK7 → SAPK signaling cascade in response to DNA damage.

The cell death machinery governed by the p53 tumor suppressor in response to DNA damage.

The cellular response to genotoxic stress that damages DNA includes cell cycle arrest, activation of DNA repair, and in the event of irreparable damage, induction of apoptosis. However, the signals that determine cell fate, that is, survival or apoptosis, are largely unclear. The tumor suppressor p53 has been implicated in many important cellular processes, including regulation of apoptotic cell death. When cells encounter genotoxic stress, certain sensors for DNA lesions eventually stabilize and activate p53. Subsequently, p53 exerts its tumor suppressor function by transactivating numerous target genes. Active p53 is subjected to a complex and diverse array of covalent post‐translational modifications, which selectively influence the expression of p53 target genes. In this regard, the molecular basis for how p53 induces apoptosis has been extensively studied; however, the relative contribution of each downstream effecter is still to be explored. Moreover, little is known about precise mechanisms by which modified p53 is capable of apoptosis induction. A thorough understanding for the whole picture of p53 modification in apoptosis will be extremely valuable in the development of highly effective and specific therapies for caner patients. This review is focused on the current views regarding the regulation of cell fate by p53 in the apoptotic response to DNA damage.

DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells

Dysregulation of the G(1)/S transition in the cell cycle contributes to tumor development. The oncogenic transcription factors c-Jun and c-Myc are indispensable regulators at this transition, and their aberrant expression is associated with many malignancies. Degradation of c-Jun/c-Myc is a critical process for the G(1)/S transition, which is initiated upon phosphorylation by glycogen synthase kinase 3 β (GSK3β). However, a specific kinase or kinases responsible for priming phosphorylation events that precede this GSK3β modification has not been definitively identified. Here, we found that the dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 functions as a priming kinase of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G(1) phase and accelerated cell proliferation due to escape of c-Jun and c-Myc from ubiquitination-mediated degradation. In concert with these results, silencing DYRK2 increased cell proliferation in human cancer cells, and this promotion was completely impeded by codeprivation of c-Jun or c-Myc in vivo. We also found marked attenuation of DYRK2 expression in multiple human tumor samples. Downregulation of DYRK2 correlated with high levels of unphosphorylated c-Jun and c-Myc and, importantly, with invasiveness of human breast cancers. These results reveal that DYRK2 regulates tumor progression through modulation of c-Jun and c-Myc.

Pim-1 controls NF- κ B signalling by stabilizing RelA/p65

Post-translational modification and degradation of proteins by the ubiquitin–proteasome system are key regulatory mechanisms in cellular responses to various stimuli. The NF-κB signaling pathway is controlled by the ubiquitin-mediated proteolysis. RelA/p65, which is a main subunit of NF-κB, is ubiquitinated for degradation by SOCS-1, but the functional mechanism of its ubiquitination remains poorly understood. In this study we show that phosphorylation of RelA/p65 at Ser276 prevents its degradation by ubiquitin-mediated proteolysis. In contrast, impairment of Ser276 phosphorylation affects constitutive degradation of RelA/p65. Importantly, we identify Pim-1 as a further kinase responsible for the phosphorylation of RelA/p65 at Ser276. Depletion of Pim-1 hinders not only Ser276 phosphorylation but also transactivation of RelA/p65 target genes. We also show that Pim-1 contributes to recruitment of RelA/p65 to κB-elements to activate NF-κB signalling after TNF-α stimulation. In concert with these results, the knockdown of Pim-1 impairs IL-6 production and augments apoptosis by interfering RelA/p65 activation. These findings provide a model in which Pim-1 phosphorylation of RelA/p65 at Ser276 allows defense against ubiquitin-mediated degradation and whereby exerts activation of NF-κB signalling.

Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells.

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of the stress-activated protein kinase (SAPK) and induction of terminal monocytic differentiation. The present studies demonstrate that TPA targets SAPK to mitochondria by a mechanism dependent on activation of protein kinase C (PKC) β. Translocation of SAPK to mitochondria in response to TPA is associated with release of cytochrome c, caspase-3 activation and induction of apoptosis. The results show that TPA induces the association of SAPK with the mitochondrial anti-apoptotic Bcl-xL protein. Overexpression of Bcl-xL attenuated the apoptotic response to TPA treatment. Moreover, expression of Bcl-xL mutated at sites of SAPK phosphorylation (Thr-47, -115) was more effective than wild-type Bcl-xL in abrogating TPA-induced cytochrome c release and apoptosis. By contrast, expression of Bcl-xL had little effect on induction of the monocytic phenotype. These findings indicate that myeloid leukemia cells respond to TPA with targeting of SAPK to mitochondria and that this response contributes to terminal differentiation through the release of cytochrome c and induction of apoptosis. Cell Death and Differentiation (2001) 8, 794–800