• Bulletin of the Korean Chemical Society
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• v.27 no.9
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• pp.1353-1358
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• 2006

We investigated in vitro T-cell inhibitory activity and bioavailability of small chemical inhibitors for Lck SH2 domain, which had a different scaffold such as an amide bond, reduced amide bond, N-methyl amide bond, thioamide bond, and urethane bond. Each of these compounds, with its particular scaffold, showed a different logP value, stability against serum enzyme, stability in buffer solution, and in vitro T-cell inhibitory activity. Overall results indicated that the SH2 inhibitor containing urethane bond can be a new lead compound because of its superior bioavailability, potent in vitro T-cell inhibitory activity, and facile synthesis.

• Development and Reproduction
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• v.2 no.2
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• pp.165-171
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• 1998

p62 is a novel cytoplsmic protein that binds to SH2 domain of p56$^{lck}$, lymphocyte-specific protein tyrosine kinase, and the expression of p62 was observed in most tissues. In addition p62 interacts with various proteins including ubiquitin and atypical PKC isoform, indicating its diverse biological role in different tissues. However, little is known about functional connection between p62 and its binding proteins. In the present study, a novel cellular protein, p62 has been shown to bind to 14-3-3 $\tau$ isoform that is specific for T cells. Moreover, overexpression of p62 in T cells caused to delay onset of UV-induced apoptosis characterized by DNA fragmentation and breakdown of poly (ADP-ribose) polymerase (PARP). Lately, 14-3-3 proteins have been shown to mediate survival signal via interacting proapoptotic Bad protein in the Iymphocyte. These results suggested the presence of p62-mediated regulatory mechanism during apoptosis in T cells, in which activation-induced apoptotic signal could be interfered by p62 and 14-3-3 protein.n.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• Molecules and Cells
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• v.43 no.11
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• pp.921-934
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• 2020

Lck-interacting transmembrane adaptor 1 (LIME) has been previously identified as a raft-associated transmembrane protein expressed predominantly in T and B lymphocytes. Although LIME is shown to transduce the immunoreceptor signaling and immunological synapse formation via its tyrosine phosphorylation by Lck, a Src-family kinase, the in vivo function of LIME has remained elusive in the previous studies. Here we report that LIME is preferentially expressed in effector T cells and mediates chemokine-mediated T cell migration. Interestingly, in LIME^-/- mice, while T cell receptor stimulation-dependent proliferation, differentiation to effector T cells, cytotoxic T lymphocyte (CTL) function and regulatory T lymphocyte (Treg) function were normal, only T cell-mediated inflammatory response was significantly defective. The reduced inflammation was accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME^-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME^-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites.

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.151-151
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• 2005

• Molecules and Cells
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• v.28 no.3
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• pp.183-188
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• 2009

TSAd/Lad is a T cell adaptor molecule involved in $p56^{lck}$-mediated T cell activation. To investigate the functions of TSAd in T cells, we generated transgenic (TG) mice expressing the SH2 domain of TSAd (TSAd-SH2) under the control of the $p56^{lck}$ proximal promoter. In T cells from TSAd-SH2 TG mice, T cell receptor (TCR)-mediated early signaling events, such as $Ca^{2+}$ flux and ERK activation, were normal; however, late activation events, such as IL-2 production and proliferation, were significantly reduced. Moreover, TCR-induced cell adhesion to extracellular matrix (ECM) proteins and migration through ECM proteins were defective in T cells from TSAd-SH2 TG mice. Furthermore, the contact hypersensitivity (CHS) reaction, an inflammatory response mainly mediated by T helper 1 (Th1) cells, was inhibited in TSAd-SH2 TG mice. Taken together, these results show that TSAd, particularly the SH2 domain of TSAd, is essential for the effector functions of T cells.

• BMB Reports
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• v.28 no.4
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4352-4360
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• 2011

A four dimensional quantitative structure activity relationship analysis was applied to a series of 50 flavonoid inhibitors of $p56^{lck}$ protein tyrosine kinase by the molecular comparative electron topological method. It was found that the -log (IC50) values of the compounds were highly dependent on the topology, size and electrostatic character of the substituents at seven positions of the flavonoid scaffold in this study. Depending on the negative or positive charge of the groups correctly embedded in these substituents, three-dimensional bio-structure to increase or decrease -log (IC50) values in the training set of 39 compounds was predicted. The test set of 11 compounds was used to evaluate the predictivity of the model. To generate 4D-QSAR model, the defined function groups and pharmacophore used as topological descriptors in the calculation of activity were of sufficient statistical quality ($R^2$ = 0.72 and $Q^2$ = 0.69). Ligand docking approach by using Dock 6.0. These compounds include many flavonoid analogs, They were docked onto human families of p56lck PTKs retrieved from the Protein Data Bank, 1lkl.pdb.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.490-496
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• 1996

Naphtodianthrone hypericin produced a potent antitumor activity in vitro against several tumor cells. However, it did not show any cytotoxicity on normal cells such as Macaccus rheus monkey kidney cells (MA-104) and primary cultured rat hepatocytes up to $500{\mu}M$ concentration. Hypericin added to A431 human epidermoid carcinoma cell membrane inhibited the autophosphorylation of the epidermal growth factor (EGF) receptor and the tyrosine phosphorylation of RR-SRC peptide catalyzed by an EGF-receptor. Similarly, treatment of the A431 cells with hypericin inhibited the tyrosine phosphorylation of EGF-dependent endogenous EGF-receptor by western blotting analysis. Hypericin also inhibited the T cell PTK, $P56^{lck}$, in a dose-dependent fashion with an $IC_{50}=5{\mu}M$. The tyrosine phosphorylation, on RR-SRC peptide and EGF-induced receptor autophosphorylation, either in vitro or in intact cells was inhibited by hypericin at the same concentration as that in A431 cell proliferation. These data suggest that hypericin directly inhibits EGF-receptor and $P56^{lck}$ PTK activity in vitro and can mediate such action in vivo.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.260.2-260
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• 1996

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