T Cell에서의 Lectin에 의한 Phospholipase $C-{\gamma}1$ Tyrosine 잔기 인산화를 통한 Phospholipase C의 활성화

김희숙,문경호,이영한,윤두희,류성호,서판길,Kim, Hee-Sook,Moon, Kyoung-Ho,Lee, Young-Han,Yun, Doo-Hee,Ryu, Sung-Ho,Suh, Pann-Ghill 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.8

사람의 $CD4^+$ T임파구 암세포인 Jurkat cell에는 phosphatidyl inositide-specific phospholipase C(PI-PLC 또는 PLC)의 이성화효소인 PLC-${\beta}1$, $-{\gamma}1$, $-{\delta}1$이 존재한다. Jurkat cell에 여러 lectin들을 처리하여 $PLC-{\gamma}1$의 인산화반응, tyrosine잔기 인산화반응 및 PI-PLC의 활성도 등을 측정하므로써 lectin들에 의한 T세포의 선호전달에 $PLC-{\gamma}1$이 관여하는지 검토하였다. Phytohem-agglutinin(PHA), Concanavalin A(Con A), Trichosanthis radix aggutinin(TRA) 등의 lectin을 T세포에 처리하였을 때 PLC의 활성에 의하여 세포의 막지질성분인 phosphatidyl inositide의 가수분해물인 inositol phosphate들의 축적이 증가하였다. 그중 세포신호전달에 있어 second messenger인 inositol 1,4,5-trisphosphate(IP3)는 30초 내에 이미 최고수준에 달하였다. 또한 처리 후 30초 이내에 $PLC-{\gamma}1$의 tyrosine잔기에 인산화가 일어났다. 인산화된 tyrosine잔기의 위치는 T cell의 CD3의 항체인 OKT3를 처리하거나 fibroblast에 성장인자인 PDGF 또는 EGF를 처리한 경우와 같음을 tryptic phosphopeptide mapping으로 확인하였다. 이와 같은 결과들은, T세포의 분화를 유도하는 lectin들이 T세포의 항원수용체와 CD3 복합체(TCR-CD3 Complex) 또는 표면당단백질인 CD4, CD8 등과 결합하거나 상호작용하고 있는 nonreceptor tyrosine kinase(fyn 또는 lck)를 활성화시키고 이들에 의해 phospholipase $C-{\gamma}1$의 tyrosine잔기가 인산화됨으로써 세포 외부 신호가 세포내로 전달되고 있음을 암시하고 있다. 이렇게 tyrosine잔기가 인산화되어 활성화된 phospholipase $C-{\gamma}1$은 다시 phosphatidyl inositide를 가수분해시켜 second messenger인 1,2-diacylglycerol(DG)과 inositol 1,4,5-trisphosphate(IP3)를 생성하게 하여 T세포의 분화 및 성장을 유도할 것으로 생각된다. Stimulation of the human T cell line, Jurkat, by the addition of mitogenic lectin activates phospholipase $C-{\gamma}1$($PLC-{\gamma}1$), generating two second messengers, inositol-l,4,5-trisphosphate and diacylglycerol, from phosphatidyl inositide. To investigate the effect of mitogenic lectins, Jurkat cells were treated with PHA, TRA (one of Trichosanthes radix agglutinins) or Can A. The tyrosine phosphorylation of $PLC-{\gamma}1$ occurred rapidly and reached maximum level less than 0.5 min after PHA stimulation in the presence of orthovanadate. In cells prelabeled with [$^3H$]myo-inositol, PHA quickly but persistently stimulates the formation of [$^3H$]inositol-1,4,5-trisphosphate within 0.5 min after stimulation. Two-dimensional phosphopeptide map analysis revealed that the major sites of tyrosine and serine phosphorylation in $PLC-{\gamma}1$ from stimulated Jurkat cells are the same as those in $PLC-{\gamma}1$ from Jurkat cells treated with antibody to CD3 or A43l cells with EGF. Thus, we suggests that mitogenic lectin-dependent increase in phosphoinositide hydrolysis in Jurkat cells can be occur through the phosphorylation of tyrosine residues on $PLC-{\gamma}1$ by a nonreceptor tyrosine kinase(s) coupled to the TCR-CD3 complex.

T Cell 에서의 Lectin 에 의한 Phospholipase C - γ1 Tyrosine 잔기 인산화를 통한 Phospholipase C 의 활성화

김희숙,문경호,이영한,윤두희,류성호,서판길 ( Hee Sook Kim,Kyoung Ho Moon,Young Han Lee,Doo Hee Yun,Sung Ho Ryu,Pann Ghill Suh ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

Stimulation of the human T cell line, Jurkat, by the addition of mitogenic lectin activates phospholipase C-γ1(PLC-γ1), generating two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol, from phosphatidyl inositide. To investigate the effect of mitogenic lectins, Jurkat cells were treated with PHA, TRA (one of Trichosanthes radix agglutinins) or Con A. The tyrosine phosphorylation of PLC-γ1 occurred rapidly and reached maximum level less than 0.5 min after PHA stimulation in the presence of orthovanadate. In cells prelabeled with [³H]myo-inositol, PHA quickly but persistently stimulates the formation of [³H]inositol-1,4,5-trisphosphate within 0.5 min after stimulation. Two-dimensional phosphopeptide map analysis revealed that the major sites of tyrosine and serine phosphorylation in PLC-γ1 from stimulated Jurkat cells are the same as those in PLC-γ1 from Jurkat cells treated with antibody to CD3 or A431 cells with EGF. Thus, we suggests that mitogenic lectin-dependent increase in phosphoinositide hydrolysis in Jurkat cells can be occur through the phosphorylation of tyrosine residues on PLC-γ1 by a nonreceptor tyrosine kinase(s) coupled to the TCR-CD3 complex.

분화된 HL60 세포에서 Granulocyte-Macrophage Colony-Stimulating Factor에 의한 95 kDa 단백질의 Tyrosine잔기 인산화

이영한,김정옥,민도식,김희숙,김용식,이창연,류성호,서판길,Lee, Young-Han,Kim, Jeong-Ock,Min, Do-Sik,Kim, Hee-Sook,Kim, Yong-Sik,Lee, Chang-Youn,Ryu, Sung-Ho,Suh, Pann-Ghill 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5

사람의 granulocyte-macrophage colony-stimulating factor(GM-CSF)는 세포 표면에 존재하는 특이 수용체 자극을 통하여 각종 분화단계에 있는 골수세포의 성장과 분화에 중요한 cytokine이다. HL60 세포에 분화 유도 인자인 phorbol 12-myristate 13-acetate(PMA), $1{\alpha}$,25-dihydroxyvitamine $D_3$[1,25-$(OH)_{2}VD_{3}$] 및 dimethyl sulfoxide(DMSO) 등을 전처리한 후 GM-CSF 자극에 대해 특정 분화단계에서만 활성화되는 세포내 단백질을 조사하였다. SH2-SH3 domain을 인지하는 F7-2 항체로 면역 침전하여 면역 블롯한 결과 미분화 세포와 DMSO를 처리한 세포에서는 GM-CSF 자극에 의해 tyrosine 인산화되는 단백질을 발견할 수 없었지만, PMA와 1,25-$(OH)_2VD_3$를 전처리한 세포에서는 95kDa 단백질의 tyrosine 인산화가 일어남을 관찰하였다. Kinetics 분석결과 95 kDa 단백질의 tyrosine 인산화 반응은 GM-CSF 처리 후 1분 이내에, 1,25-$(OH)_{2}VD_{3}$를 전처리한 세포에서는 2분 이내에 나타나는 신속한 반응이었다. 이 단백질이 세포에서 발현되는 양은 PMA 전처리 시간에 무관하게 일정하였으나 GM-CSF에 의한 인산화는 PMA 전처리 시간에 따라 변화하였다. 또한, 95 kDa 단백질은 in vitro에서도 autophosphorylation되었다. 이러한 결과로부터 95 kDa 단백질은 PMA나 1,25-$(OH)_{2}VD_{3}$로 분화 유도된 HL60 세포에서 GM-CSF의 초기 신호전달 경로에 관여하고 있음을 추측할 수 있었다. Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine which stimulates the proliferation and differentiation of various lineage of hematopoietic cells. We examined whether GM-CSF stimulates protein phosphorylation in HL60 cells pretreated with differentiation-inducing factors such as PMA, 1,25-$(OH)_{2}VD_{3}$ and DMSO. GM-CSF induced tyrosine phosphorylation of 95 kDa protein in PMA- or 1,25-$(OH)_{2}VD_{3}$ but not in DMSO-pretreated cells. Tyrosine phosphorylation of 95 kDa was detected at 1 min and 2 min after stimulation of GM-CSF in PMA- and 1,25-$(OH)_{2}VD_{3}$-pretreated cells, respectively. Kinase activity which phosphorylates tyrosine residue(s) of the 95 kDa protein appeared to increase in a time dependent manner in PMA-pretreated cells, whereas the expression level of 95 kDa protein was not changed. We also observed that 95 kDa protein was autophosphorylated in immunecomplex kinase assay, suggesting that this 95 kDa protein may be tyrosine kinase which is activated in lineage specific manner. These results suggest that 95 kDa protein may be involved in an early signal transduction pathway of GM-CSF.

Jurkat T 면역세포에서 Phosphoinositides 의 가수분해를 증가시키는 약용식물 추출물의 검색

민도식(Do Sik Min),이영한(Young Han Lee),백석환(Suk Hwan Baek),서판길(Pann Ghill Suh),류성호(Sung Ho Ryu) 한국응용약물학회 1996 Biomolecules & Therapeutics(구 응용약물학회지) Vol.4 No.2

Activation of the T lymphocytes results in a variety of early biochemical events ultimately leading to cell proliferation and lymphokine production. Stimulation of the signal transduction cascade in T cells through the T cell receptor coincides with activation of the phosphatidylinositol-phospholipase C (PI-PLC) pathway. Therefore, we have established a model system to screen immuno-simulator that can increase the hydrolysis of phosphoinositides in human T cell leukemia Jurkat cells. As a result of screening from herbal medicine extract, 4 extracts (Olibanum, Ephedrae Herba, Real Gar, Saussureae Radix) were found to increase the production of inositol phosphates. All the active fraction from the four kinds of extract were eluted in a different retention time on C-18 HPLC and these active fraction also showed difference in cell specificity. And all the active fractions increased DNA synthesis in T cell. Therefore, it is suggested that the active fraction among 4 extracts might contain a compound having different properties one another.

분화된 HL60 세포에서 Granulocyte - Macrophage Colony - Stimulating Factor 에 의한 95kDa 단백질의 Tyrosine 잔기 인산화

이영한,김정옥,민도식,김희숙,김용식,이창연,류성호,서판길 ( Young Han Lee,Jeong Ock Kim,Do Sik Min,Hee Sook Kim,Yong Sik Kim,Chang Youn Lee,Sung Ho Ryu,Pann Ghill Suh ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine which stimulates the proliferation and differentiation of various lineage of hematopoietic cells. We examined whether GM-CSF stimulates protein phosphorylation in HL60 cells pretreated with differentiation-inducing factors such as PMA, 1,25-(OH)₂VD₃ and DMSO. GM-CSF induced tyrosine phosphorylation of 95 kDa protein in PMA- or 1,25-(OH)₂VD₃ but not in DMSO-pretreated cells. Tyrosine phosphorylation of 95 kDa was detected at 1 min and 2 min after stimulation of GM-CSF in PMA- and 1,25-(OH)₂VD₃-pretreated cells, respectively. Kinase activity which phosphorylates tyrosine residues) of the 95 kDa protein appeared to increase in a time dependent manner in PMA-pretreated cells, whereas the expression level of 95 kDa protein was not changed. We also observed that 95 kDa protein was autophosphorylated in immunecomplex kinase assay, suggesting that this 95 kDa protein may be tyrosine kinase which is activated in lineage specific manner. These results suggest that 95 kDa protein may be involved in an early signal transduction pathway of GM-CSF.

방사선 조사 후 백서 공장 점막의 재생과정에서 5-fluorouracil 투여가 phospholipase C 와 ras 암유전자단백의 발현에 미치는 영향

박경란(Kyung Ran Park),이정식(Chung Sik Rhee),김성숙(Sung Sook Kim),이영한(Young Han Lee),류성호(Sung Ho Ryu),서판길(Pann Ghill Suh) 대한방사선종양학회 1994 Radiation Oncology Journal Vol.12 No.3

Purpose : Phospholipase C(PLC) isozymes play significant roles in transmembranesignal transduction. PLC-γ1 acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU Meterials and Methods : To determine the effect of the administrations mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group Ⅰ had no treatment. Group Ⅱ received radiation(8 Gy) only. Group Ⅲ received radiation(8Gy) and 5-FU(150mg/kg) continuous intravenous (ⅳ) infusion for 12 hours. Group Ⅳ received radiation(8Gy) and 5-FU(150mg/kg) ⅳbolus injection. Group Ⅴ received only 5-FU(150mg/kg) continuous ⅳ infusion for 12 hours. Group Ⅵ received only 5-FU(150mg/kg) ⅳ bolus injection. Through immunoblotting and immumohistochemistry, we exmained the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU adminstration. We also investigated the histological findings using hematoxyling and eosin stain. Results : In the immumohistochemistry study, PLC-γ1 expression was the highest in group Ⅲ followed by groups Ⅱ and Ⅳ in that order and was weakly positive in groups Ⅴ and Ⅵ. PLC-γ1 was hardly detected in the control group. The expression of ras oncoprotein was the same as the PLC-γ1 expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-γ1 expression was the highest in group Ⅲ followed by group Ⅳ and Ⅱ in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-γ1 on the damaged surface epithelium rather than to its expression in the regeneration region as obsrved in the immunohistochemistry study for group Ⅳ. The expression of PLC-γ1 was positive only in group Ⅴ and Ⅵ, which received both radiation and 5-FU and the expression of PLC-β1 was negligible for all groups. Conclusion : These results suggest that PLC-γ1 mediated signal transduction and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous ⅳ infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition the expression of PLC-δ1 in combined group of radiation and 5-FU implies that PLC-δ1 may be involved in signal transduction mediated by concerted action between radiation and 5-FU.

방사선과 5-fluorouracil 투여후 백서 공장 점막의 재생과정에서 Phospholipase C-γ1의 발현에 관한 면역조직화학적 연구

박경란,이종영,김성숙,고창민,정순희,이영한,서판길 연세대학교 원주의과대학 1995 원주의대논문집 Vol.8 No.1