Jae-Rin Lee

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.

Cdo Interacts with APPL1 and Activates AKT in Myoblast Differentiation

Cdo activates Akt via indirect interaction with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via interaction with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt.

Effect of Cl- on tyrosinase: complex inhibition kinetics and biochemical implication.

Abstract Tyrosinase plays a core role in melanogenesis of the various organisms. Therefore, the regulation of the tyrosinase activity is directly related with melanin synthesis. In this study, we investigated the Cl−-induced inhibition of human tyrosinase and the potent role of Cl− as a negative regulator in melanogenesis. For the inhibition kinetic studies, human tyrosinase was differently prepared from the TXM13 melanotic cells as well as from cells that had undergone gene transfection. We found that Cl− inhibited tyrosinase in a slope-parabolic competitive manner and tyrosinase gene transfection into HEK293 cell significantly down-regulated the expression levels of solute carrier family 12, member 4 (potassium/chloride transporters, SLC12A7) and solute carrier family 12, member 7 (potassium/chloride transporters, SLC12A7), which are known to be Cl− transporters. From the results of the inhibition kinetic studies and the Cl− transporter expression level, we suggested that Cl− might act as a potent regulatory factor in melanogenesis. It is worth notice that a high content of Cl− exists physiologically and tyrosinase reacts sensitively to Cl− in a complex interaction manner.

Interactions among Four Proteins Encoded by the Human Cytomegalovirus UL112-113 Region Regulate Their Intranuclear Targeting and the Recruitment of UL44 to Prereplication Foci

ABSTRACT Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.

BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression via an induction of senescence through the regulation of STAT3/SKP2/p27 pathway.

Aggregation and Folding of Recombinant Human Creatine Kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble fraction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (k1 = 6.5 × 10−3 s−1; k2 = 0.54 × 10−3 s−1) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (ΔΔG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (k1 = 3.0 × 10−3 s−1; k2 = 0.64 × 10−3 s−1). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.

PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity.

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.

PKN2 and Cdo interact to activate AKT and promote myoblast differentiation

Skeletal myogenesis is coordinated by multiple signaling pathways that control cell adhesion/migration, survival and differentiation accompanied by muscle-specific gene expression. A cell surface protein Cdo is involved in cell contact-mediated promyogenic signals through activation of p38MAPK and AKT. Protein kinase C-related kinase 2 (PKN2/PRK2) is implicated in regulation of various biological processes, including cell migration, adhesion and death. It has been shown to interact with and inhibit AKT thereby inducing cell death. This led us to investigate the role of PKN2 in skeletal myogenesis and the crosstalk between PKN2 and Cdo. Like Cdo, PKN2 was upregulated in C2C12 myoblasts during differentiation and decreased in cells with Cdo depletion caused by shRNA or cultured on integrin-independent substratum. This decline of PKN2 levels resulted in diminished AKT activation during myoblast differentiation. Consistently, PKN2 overexpression-enhanced C2C12 myoblast differentiation, whereas PKN2-depletion impaired it, without affecting cell survival. PKN2 formed complexes with Cdo, APPL1 and AKT via its C-terminal region and this interaction appeared to be important for induction of AKT activity as well as myoblast differentiation. Furthermore, PKN2-enhanced MyoD-responsive reporter activities by mediating the recruitment of BAF60c and MyoD to the myogenin promoter. Taken together, PKN2 has a critical role in cell adhesion-mediated AKT activation during myoblast differentiation.

Regulation of inflammatory gene expression in macrophages by epithelial-stromal interaction 1 (Epsti1)

Epithelial-stromal interaction 1 (EPSTI1) was first discovered as a gene induced in breast cancer epithelial cells by co-cultured stromal fibroblasts. There are many reports on the role of Epsti1 in cancer malignancy. Epsti1 is now well known in regulating cancer. Recently, the role of Epsti1 in the immune response has been reported; these reports suggest the role of Epsti1 in immune function, immune privilege, and autoimmune diseases. Furthermore, they show that Epsti1 is expressed in various types of immune cells. In this study, we observed that Epsti1 is highly expressed in macrophages exposed to IFNγ and lipopolysaccharide (LPS), which classically activates macrophages. Polarization of macrophage to classically activated (M1) or alternatively activated (M2) is important for mounting responses against various infections. The M1 and M2 types of macrophage have a distinct role in the immune system. However, the molecular mechanism of modulation of the macrophage type is not well defined. Our results showed that the M2 type macrophage phenotype is enhanced in Epsti1-deficient bone marrow-derived macrophages (BMDM). In addition, Epsti1 deficiency suppresses induction of pro-inflammatory genes in BMDMs via inhibition of Stat1 and p65 nuclear localization and phosphorylation. Surprisingly, Epsti1-/- mice show decreased numbers of M1 macrophages in the peritoneal cavity. These findings identify Epsti1 as a modulator of macrophage activation and polarization via the Stat1 and p65 pathways, and suggest a potentially important role of Epsti1 in immunotherapies against inflammatory diseases.

Bisphenol A and estradiol impede myoblast differentiation through down-regulating Akt signaling pathway

Bisphenol A (BPA), one of the most widespread endocrine disrupting chemicals, is known as an artificial estrogen, which interacts with estrogen receptor (ER). In this study, we investigated the effects of BPA and estradiol on myoblast differentiation and the underlying signaling mechanism. Exposure to BPA (0.01-1 μM) in mouse myoblast C2C12 cells attenuated myogenic differentiation via the reduced expression of muscle-specific genes, such as myosin heavy chain (MHC), MyoD, and Myogenin, without the alteration of cell proliferation and viability. BPA-exposed C2C12 myoblasts also showed a reduction of Akt phosphorylation ((37-61) %, p < 0.001), a key event for myogenesis. Similarly to BPA, estradiol (0.01-1 μM) reduced the expression of muscle-specific proteins and the formation of multinucleated myotubes, and attenuated the muscle differentiation-specific phosphorylation of Akt ((42-59) %, p < 0.001). We conclude that BPA and estradiol suppress myogenic differentiation through the inhibition of Akt signaling.