Myong-Joon Hahn

Differential activation of MAP kinase family members triggered by CD99 engagement

The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti‐CD99 antibody led to activation of three mitogen‐activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI‐3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca2+.

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.

Ubiquitin specific protease 4 positively regulates the WNT/β-catenin signaling in colorectal cancer

β‐catenin is a key signal transducer in the canonical WNT pathway and is negatively regulated by ubiquitin‐dependent proteolysis. Through screening of various deubiquitinating enzymes (DUBs), we identified ubiquitin specific protease 4 (USP4) as a candidate for β‐catenin‐specific DUB. The effects of USP4 overexpression or knockdown suggested that USP4 positively controls the stability of β‐catenin and enhances β‐catenin‐regulated transcription. Domain mapping results revealed that the C‐terminal catalytic domain is responsible for β‐catenin binding and nuclear transport. Examination of colon cancer tissues from patients revealed a correlation between elevated expression levels of USP4 and β‐catenin. Consistent with this correlation, USP4 knockdown in HCT116, a colon cancer cell line, reduced invasion and migration activity. These observations indicate that USP4 acts as a positive regulator of the WNT/β‐catenin pathway by deubiquitination and facilitates nuclear localization of β‐catenin. Therefore, we propose that USP4 is a potential target for anti‐cancer therapeutics.

Complex Inhibition of Tyrosinase by Thiol-Composed Cu2+ Chelators: A Clue for Designing Whitening Agents

Abstract The inhibition of tyrosinase has attracted considerable attention for potential medicinal and cosmetic applications, as well as in agriculture. This study investigated the inhibition effects of thiol-associated Cu2+ chelators and deduced a strategy for designing and/or selecting tyrosinase inhibitors. Among the several compounds tested, dithioglycerine (DTGC) was selected for further experiments on the inhibition kinetics on tyrosinase. Different types of tyrosinases derived from mushroom and from the transient overexpression in HEK293 cells were tested individually. The results showed that DTGC significantly inhibited human tyrosinase in a complex manner (slope-parabolic mixed-type inhibition), which was comparable to mushroom tyrosinase. The affinity of DTGC affinity to human tyrosinase was evaluated by setting up a Ki slope equation. The results suggest that a Cu2+ chelator modified with thiol groups has potential as a whitening agent. In addition, a strategy for designing and/or selecting tyrosinase inhibitors that target the active enzyme site was also suggested.

Towards Profiling the Gene Expression of Tyrosinase-induced Melanogenesis in HEK293 Cells: a Functional DNA Chip Microarray and Interactomics Studies

Abstract The overexpression of a single tyrosinase gene can induce conspicuous pigmentation in nonpigmented cells. We hypothesized that some unknown tyrosinase-associated genes are simultaneously regulated by melanin synthesis. To improve understanding of melanogenesis and tyrosinase-associated functions, we attempted to profile the genes that are altered during melanin production in HEK293 cells by using a functional DNA chip microarray. The candidate genes were obtained based on significance analysis of microarray (SAM) and further computational prediction via protein-protein interaction (PPI) mapping suggested that newly detected hub genes were involved in melanogenesis. PPI mapping using bioinformatic tools revealed 8 genes that formed an interaction hub. The yeast two-hybridization results suggested some candidate genes could interact with tyrosinase. The present study provides information to further understand the complex factors associated with tyrosinase- induced melanogenesis and apoptosis. The approach of combining expression data analysis and predicted protein interaction partners can help identify genes involved in pigmentation.

Requirement of MEF2D in the induced differentiation of HL60 promyeloid cells

The regulatory role of MEF2 (myocyte enhancer binding factor 2) proteins in nonmuscle tissues has not been well characterized. We examined the expression of MEF2 family members, namely, MEF2A, -B, -C, and -D, in the differentiation of HL60 promyeloid cells and observed the remarkable increase in the expressions of MEF2A and MEF2D proteins during the differentiation process into monocytes. To examine the role of MEF2, we expressed a dominant-negative form of MEF2D, without its transactivation domain, in HL60 cells. When the HL60 cell line expressing the mutant MEF2D was induced to differentiate by VitD(3) treatment, cell surface expression of CD14 and the ability to reduce NBT, which are important characteristics of differentiated monocytes, were significantly decreased compared with control HL60 cells. These results show that MEF2D is required in the differentiation process along the monocyte/macrophage lineage,

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.

Effect of Thiohydroxyl Compounds on Tyrosinase: Inactivation and Reactivation Study

An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and β-mercaptoethanol (β-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and β-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dosedependent manner. Sequential kinetic studies revealed that DTT and β-ME caused different mixed-type inhibition mechanisms: the slope–parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope–hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by β-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and β-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and β-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.