Biomedical subjects

Keiko Morotomi-Yano

Publications and source records attributed to Keiko Morotomi-Yano.

4 recordsLinked to original sources

PubMed2005

Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks.

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.

Amino Acid Sequence↗

PubMed2004

DNA-PKcs function regulated specifically by protein phosphatase 5.

Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.

Animals↗

PubMed2002

Identification and characterization of the human protein kinase-like gene NTKL: mitosis-specific centrosomal localization of an alternatively spliced isoform.

Although the centrosome has an essential role in mitosis, its molecular components have not been fully elucidated. Here, we describe the molecular cloning and characterization of the human gene NTKL, which encodes an evolutionarily conserved kinase-like protein. NTKL mRNA is found ubiquitously in human tissues. NTKL is located on 11q13 and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Alternative splicing generates two variant forms of NTKL mRNA that encode protein isoforms with internal deletions. When fused to green fluorescent protein, the full-length product and one of the variant proteins are found in cytoplasm. The other variant product also exists in the cytoplasm during interphase, but is found in the centrosomes during mitosis. Endogenous NTKL protein is also localized to the centrosomes during mitosis. This cell-cycle-dependent centrosomal localization suggests that NTKL is involved in centrosome-related cellular functions.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Human regulatory factor X 4 (RFX4) is a testis-specific dimeric DNA-binding protein that cooperates with other human RFX members.

Regulatory factor X (RFX) members are evolutionarily conserved transcription factors that share a highly conserved winged helix DNA-binding domain. Human RFX4 has been isolated from breast cancer as a partial cDNA encoding a short RFX-type DNA-binding domain fused to the estrogen receptor, but the entire structure of RFX4 has been unknown. Here, we report the molecular cloning and characterization of human RFX4. RFX4 contains evolutionarily conserved regions, including a RFX-type DNA-binding domain, a dimerization domain, and other conserved regions, and is closely related to RFX1, RFX2, and RFX3 in structure. The expression of RFX4 is restricted to testis. In vitro synthesized RFX4 protein bound to typical RFX binding sites in a sequence-dependent manner. Immunoprecipitation analyses showed that RFX4 interacts physically with RFX2, RFX3, and RFX4 itself but not with RFX1. In contrast to other mammalian RFX members that form dimers, RFX4 is revealed to have no distinct transcriptional activation domains. By using a chimeric protein of RFX1 and RFX4, the C-terminal domain of RFX4 was shown to be a possible transcriptional repression domain. Taken together, these results indicate that RFX4 is the first mammalian member of RFX family without transcriptional activation capacity and might function through selective interactions with other RFX members in transcriptional regulation.

Amino Acid Sequence↗