• BMB Reports
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• 제31권4호
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• BMB Reports
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• 제41권8호
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• pp.575-580
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• 2008

Thymocyte-specific transcriptional regulatory systems can be used to better understand the relationship between transcription and V(D)J recombination during early T cell development. In this study, we generated transgenic mice expressing the transactivator Gal4-VP16 or the Gal4 DNA binding domain (Gal4-DBD) under the control of the lck proximal promoter, which is only active in immature thymocytes. From these studies Gal4-VP16 and Gal4-DBD expression was shown to significantly alter thymic cellularity and differentiation without significantly changing the $CD3^+$ thymocyte distribution. Furthermore, the presence of Gal4-VP16 or Gal4-DBD in the transgenic thymocytes retarded the mobility of the Gal4 DNA binding motif as determined by a gel mobility shift assay, suggesting that the developmental alteration did not affect the functional property of the transgenic proteins. These results indicated that lck promoter-driven Gal4-VP16 or Gal4-DBD expression did not affect $CD3^+$ mature thymocytes, thus this system can be applied to study transcriptional regulation of transresponder genes in bigenic mouse model thymocytes.

• Bulletin of the Korean Chemical Society
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• 제28권2호
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• pp.200-206
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• 2007

Pharmacophore models for lymphocyte-specific protein tyrosine kinase (P56 LCK) were developed using CATALYST HypoGen with a training set comprising of 25 different P56 LCK inhibitors. The best quantitative pharmacophore hypothesis comprises of one hydrogen bond acceptor, one hydrogen bond donor, one hydrophobic aliphatic and one ring aromatic features with correlation coefficient of 0.941, root mean square deviation (RMSD) of 0.933 and cost difference (null cost-total cost) of 66.23. The pharmacophore model was validated by two methods and the validated model was further used to search databases for new compounds with good estimated LCK inhibitory activity. These compounds were evaluated for their binding properties at the active site by molecular docking studies using GOLD software. The compounds with good estimated activity and docking scores were evaluated for physiological properties based on Lipinski's rules. Finally 68 compounds satisfied all the properties required to be a successful inhibitor candidate.

• BMB Reports
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• 제34권6호
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.165-171
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• 1998

p62는 임파구에 특이적으로 발현하는 단백질 티로신 키나제인 p56$^{lck}$의 SH2 doamin과 결합하는 세포질 단백질로서 두 단백질의 결합에는 지금까지 알려진 바와 다르게 인산화된 티로신이 필요없다. p62는 기능이 다른 여러 조직에서 공통적으로 발현되며 유비퀴틴, 단백질 키나제 C 이성질체 둥 다양한 단백질과 결합하는 것이 알려져 있다. 이와 같은 현상으로 p62가 다양한 생물학적 기능을 수행할 수 있음을 예측할 수 있으나 그 자세한 기작은 잘 알려져 있지 않다. 본 연구에서는 p62가 T-세포에 특이적으로 발현하는 14-3-3 $ au$ 이성질체와 결합하는 것을 확인하였으며, p62를 인위적으로 T-세포에 다량으로 발현시키면 세포예정사 (apoptosis)의 시작이 지연되는 현상을 조사하였다. 이때 세포사멸과정에서 전형적으로 나타나는 DNA 절단현상 (DNA fragmentaion)과 poly (ADP-ribose) polymerase의 분해가 지연됨을 알 수 있었다. 최근 14-3-3 단백질이 임파구에서 세포예정사를 촉진시키는 기능을 가진 Bad와 결합함으로써 세포의 생존 신호 전달에 중요한 역할을 한다는 것이 보고된 바 있다. 따라서 본 연구의 결과는 T-세포의 활성으로 일어나는 사멸예정사 과정 중에 p62와 14-3-3 단백질에 의해 수행되는 조절 기작이 있음을 시사하고 있다.다.

• 한국산학기술학회논문지
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• 제20권9호
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• pp.211-226
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• 2019

백혈구 공통 항원인 돼지 CD45는 PTPRC 유전자에 암호화 되어 있으며, CD45엑손의 선택적 스플라이싱에 따라 다른 T-세포에서 발현되는 티로신 인산분해효소이다. CD45는 기질인 TCR의 $CD3{\zeta}$ 사슬, Lck, Fyn, Zap-70 kinase의 인산화된 티로신에서 인산을 분해하여 T-세포 항원 수용체(TCR) 매개 신호전달을 조절한다. CD45의 조절이상은 많은 면역 질환과 관련이 있어서, CD45는 면역약물 개발에 표적이 되어왔다. TCR 신호전달의 조절효과를 가진 주요 구조적 특징을 특성화하기 위해, 사람의 알려진 CD45 구조를 템플릿으로 적용하여 돼지 CD45RO(가장 작은 CD45 isoform)의 단백질 구조와 예측된 돼지 CD45RO 모델구조에 $CD3{\zeta}$ 사슬의 ITAM(REEpYDV)를 도킹하여 CD45RO/ITAM 펩타이드 결합구조를 예측하였다. 돼지 CD45RO의 구조적 특징은 세포외영역의 구조견고성과 세포질 내 티로신 인산분해효소 도메인의 KNRY와 PTP signature 기능모티프(두 기능 모티프는 ITAM 펩타이드 결합부위의 좁은 입구로 역할)에 있었다. 주요 구조특성은 돼지 CD45RO-ITAM 펩타이드 결합구조 안정성과 결합친화력을 조절하면서 기질 선택성에 영향을 준다. 돼지 CD45RO의 구조적 특성은 T-세포에 특이적인 면역 조절제를 탐색하는 데에 적용될 것이다.

• Animal cells and systems
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• 제3권3호
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• pp.331-336
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• 1999

p56$^{Ick}$, a cytoplasmic protein tyrosine kinase of the src family, is non-covalently associated with the cell surface coreceptors CD4 and CD8, which are expressed on thymocytes and mature T cells. The coreceptor protein plays an important role during the differentiation of thymocytes and the activation of T cells. DNA constructs were designed to study the roles of CD4 and CD8 during the differentiation of thymocytes. One is a chimeric cDNA which consists of coding regions for the extracellular domain of CD8a and the transmembrane and cytoplasmic domain of CD4. The other is the same chimeric cDNA but with a point mutation converting Cys to Ala in the Ick-binding site to disrupt the association. We confirmed that the CD8a/CD4 chimeric molecule bound to Ick more efficiently than the wild type CD8a protein. However, the chimeric protein with the Cys$leftrightarro$Ala mutation did not associate with Ick. The results suggest a possibility that the CD8a/CD4 chimeric protein may behave like a CD4 protein in associating with Ick and that it may deliver a signal inside the cell in a similar manner, Analysing effects of the mutant CD8a/CD4 chimeric protein expression in developing thymocytes will elucidate the role of Ick during the determination of CD4/CD8 cell lineages.