Biomedical subjects

Seong-Tae Kim

Publications and source records attributed to Seong-Tae Kim.

13 recordsLinked to original sources

PubMed2006

Different roles of histone H3 lysine 4 methylation in chromatin maintenance.

Histone H3 methyltransferases are involved in the epigenetic control of transcription and heterochromatin maintenance. In Saccharomyces cerevisiae, deletion of a histone H3 methyltransferase SET1 leads to the induction of a subset of stress responsive genes in a Rad53 dependent manner. This type of induction was observed only in the absence of SET1 and not in the absence of other histone methyltransferases, SET2 or DOT1. We show that the increased expression of the stress responsive genes results from a lack of histone H3 lysine (K) 4 methylation. The loss of mono-methylation on H3 K4 is necessary to increase the expression of the stress responsive genes, while the loss of di- or tri-methylation induced by deletion of either RRM domain of Set1 or the upstream effector molecules hardly affected their expression. These results suggest that mono- and multiple methylation of H3 K4 have different roles. The mono-methylation of H3 K4 might be required for the global integrity of chromatin structure, which is normally monitored by the Rad53 dependent chromatin surveillance system.

Cell Cycle Proteins↗

PubMed2006

Direct interaction between cohesin complex and DNA replication machinery.

Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.

Cell Cycle Proteins↗

PubMed2006

Arthroscopic anterior cruciate ligament reconstruction with the tibial-remnant preserving technique using a hamstring graft.

We propose that the tibial remnant of the anterior cruciate ligament (ACL) is able to enhance the revascularization and cellular proliferation of the graft, to preserve proprioceptive function, and to be able to acquire anatomic placement of the graft without roof impingement. Therefore, it seems reasonable to assume that preserving the tibial remnant as much as possible as a source of reinnervation, if technically possible without causing impingement, would be of potential benefit to the patient. Our surgical technique was developed to maximize the preservation of the tibial remnant. The distally attached semitendinosus and gracilis tendons are harvested using the tendon stripper. After satisfactory placement of 2 guide pins convergently, a closed-end socket in the lateral femoral condyle is created using an adequately sized curved curette. For anatomic placement of the graft, the tibial tunnel should be positioned within the boundaries of the normal ACL tibial remnant. The reamer must be advanced very carefully to minimize injury to the residual remnant at the intra-articular margin of the tibial tunnel. Penetration should stop at the base of the stump. The folded grafts are then pulled intra-articularly through the tibial tunnel, the tibial remnant, and the femoral socket by pulling sutures under arthroscopic visualization. The ACL tibial remnant is compacted by the tendon passage. The graft is secured proximally by tying sutures in the lateral femoral condyle and distally at the tibia with double staples by a belt-buckle method. The advantages of our technique include maximal preservation of the tibial remnant, no roof impingement caused by intrasynovial anatomic placement of the graft, the simplicity of the procedure, the minimal need for hardware or special instruments, the economic benefit, and the potential prevention of tibial tunnel enlargement by preventing synovial fluid leakage.

Anterior Cruciate Ligament↗

PubMed2005

Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649.

The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function.

Amino Acid Sequence↗

PubMed2005

Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution.

The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.

Amino Acid Sequence↗

PubMed2005

Requirement of the coiled-coil domain of PML-RARalpha oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation.

Homo-oligomerization via a coiled-coil (C-C) domain has been shown to be necessary for the promyelocytic leukemia (PML)-retinoic acid receptor-alpha (RARalpha) fusion protein to acquire oncogenic potential in acute promyelocytic leukemia. We show here that PML(DeltaC-C)-RARalpha, which contains a deletion in its C-C domain, is neither localized as characteristic microspeckles nor modified by small ubiquitin-like modifiers (SUMO). The absence of sumoylation of the DeltaC-C mutant was due to the lack of binding to Ubc9, a SUMO conjugation enzyme. The integrity of RING finger domain was also needed for both sumoylation and microspeckle formation. In GAL4-DNA tethering assays, the DeltaC-C mutant completely lost the inhibitory effect on retinoic acid (RA)-mediated transactivation. Furthermore, the expression of CD14 in U937 cells expressing the DeltaC-C mutant in response to vitamin D3 was markedly higher than in cells expressing PML-RARalpha. However, the RA-mediated induction of C/EBPbeta in cells expressing the DeltaC-C mutant was comparable to that of control cells. Thus, our results suggest that the C-C domain-associated functions of sumoylation, localization as microspeckles, and the inhibition of monocyte differentiation all contribute to the oncogenic activity of PML-RARalpha.

Active Transport, Cell Nucleus↗

PubMed2005

CtBP represses p300-mediated transcriptional activation by direct association with its bromodomain.

Histone acetyltransferase coactivators bind to acetylated histones through their bromodomains and catalyze the acetylation of histone H3 and H4 tails for transcriptional activation. C-terminal binding protein (CtBP) serves as a transcriptional corepressor by recruiting histone deacetylases. However, the precise mechanism by which CtBP represses transcription has not been determined. In this study we found that CtBP1 directly associates with p300 by binding to the PXDLS motif in the bromodomain of p300. Moreover, CtBP1 blocks the accessibility of p300 to histones in an NADH-sensitive manner and thus represses p300-mediated histone acetylation and transcriptional activation. In addition, an NADH-nonresponsive, monomeric mutant, CtBP1 (G183V), was found to strongly repress p300-mediated transcriptional activation. Thus, the dissociation of NADH from CtBP1 leads to the repression of p300-driven general transcriptional activity by CtBP1. These results suggest a novel mechanism whereby CtBP1 serves as an energy-sensing repressor of histone acetyltransferase(s) and thus affects general transcription.

Acetyltransferases↗

PubMed2004

mRNA capping enzyme activity is coupled to an early transcription elongation.

One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.

Alleles↗

PubMed2003

Determination of substrate specificity and putative substrates of Chk2 kinase.

Chk2/hCds1, the human homolog of Saccharomyces cerevisiae Rad53p and Schizosaccharomyces pombe Cds1p, plays a critical role in the DNA damage checkpoint pathway. While several in vivo targets of Chk2 have been identified, the other target proteins of Chk2 responsible for multiple functions, such as cell cycle arrest, DNA repair, and apoptosis, remain to be elucidated. We utilized the GST-peptide approach to identify physiological substrates for Chk2. Mutational analyses using GST-linked Cdc25A containing serine 123 revealed that residues at positions -5 and -3 are critical determinants for the recognition of the Chk2 substrate. We determined the general phosphorylation consensus sequence and identified in vitro targets of Chk2 using GST peptides as substrates. The newly identified in vitro target proteins include Abl1, Bub1R, Bub1, Bub3, Psk-H1, Smc3, Plk1, Cdc25B, Dcamkl1, Mre11, Pms1, and Xrcc9.

Amino Acid Sequence↗

PubMed2002

Phosphorylation of serine 1387 in Brca1 is specifically required for the Atm-mediated S-phase checkpoint after ionizing irradiation.

Although it is well established that inheritance of mutations in the Brca1 gene significantly increases the chances of developing breast or ovarian cancers, the mechanisms underlying this specific tumor susceptibility remain to be clarified. It is clear that one of the roles of the Brca1 protein is to facilitate cellular responses to DNA damage. We recently reported that Brca1 function is required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. We also found that mutation of serine 1423 in Brca1, a target of Atm phosphorylation, abrogates the G2-M checkpoint but not the ionizing irradiation-induced S-phase checkpoint. Here we demonstrate that mutation of serine 1387 in Brca1, another target of Atm phosphorylation, conversely abrogates the radiation-induced S-phase arrest but does not affect the G2-M checkpoint. Thus, these two posttranslational modifications of Brca1 have two distinct functional roles in the protein. In addition, although mutation of this site abrogates the ionizing irradiation-induced S-phase arrest, it does not adversely affect cell survival after irradiation. This demonstrates that loss of this checkpoint function by itself does not affect cell survival and suggests that some other function of Brca1 alters cell survival after DNA damage.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2002

Convergence of the fanconi anemia and ataxia telangiectasia signaling pathways.

Fanconi anemia (FA) and ataxia telangiectasia (AT) are clinically distinct autosomal recessive disorders characterized by spontaneous chromosome breakage and hematological cancers. FA cells are hypersensitive to mitomycin C (MMC), while AT cells are hypersensitive to ionizing radiation (IR). Here, we identify the Fanconi anemia protein, FANCD2, as a link between the FA and ATM damage response pathways. ATM phosphorylates FANCD2 on serine 222 in vitro. This site is also phosphorylated in vivo in an ATM-dependent manner following IR. Phosphorylation of FANCD2 is required for activation of an S phase checkpoint. The ATM-dependent phosphorylation of FANCD2 on S222 and the FA pathway-dependent monoubiquitination of FANCD2 on K561 are independent posttranslational modifications regulating discrete cellular signaling pathways. Biallelic disruption of FANCD2 results in both MMC and IR hypersensitivity.

Ataxia Telangiectasia↗

PubMed2002

Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage.

Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. Here, we show that the protein kinase, Atm, which belongs to a family of phosphatidylinositol 3-kinases that regulate cell cycle checkpoints and DNA recombination and repair, phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2002

Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation.

Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G(1), S, and G(2) phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G(2) checkpoint and a prolonged G(2) arrest after IR, suggesting the existence of different types of G(2) arrest. Two molecularly distinct G(2)/M checkpoints were identified, and the critical importance of the choice of G(2)/M checkpoint assay was demonstrated. The first of these G(2)/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G(2) at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G(2) cells must be used to assess this arrest. In contrast, G(2)/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G(2)/M accumulation after IR is not affected by the early G(2)/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G(2) arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G(2) checkpoints appear to affect survival following irradiation. Thus, two different G(2) arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.

Ataxia Telangiectasia↗