Symposium 2 : Cell Signaling, Growth and Death ; Ras activation mediated by phospholipase C-γ1

서판길 ( Pann Ghill Suh ) 한국생화학분자생물학회 (구 한국생화학회) 2000 추계학술대회집 Vol.2000 No.-

Symposia / S8-3 : Proteolytic cleavage of phospholipase C-γ1 during apoptosis

서판길(Pann Ghill Suh),(Sun Sik Bae),(Sung Ho Ryu) 한국생화학분자생물학회 (구 한국생화학회) 1999 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Phospholipase C-γ Activation by Direct Interaction with β-Tubulin Isotypes

이인범,김성국,최장현,서판길,장종수,Lee, In-Bum,Kim, Sung-Kuk,Choi, Jang-Hyun,Suh, Pann-Ghill,Chang, Jong-Soo Korean Society of Life Science 2006 생명과학회지 Vol.16 No.4

Phosphoinositide-specific phospholipase $C-{\gamma}\;1\; (PLC-{\gamma}\;1)$ has pivotal roles in cellular signaling by producing second messengers, inositol 1,4,5-trisphosphate $(IP_3)$ and diacylglycerol (DG). Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of ${\alpha}-$ and ${\beta}-tubulin$ heterodimers in all eukaryotic cells. In humans, six ${\beta}-tubulin$ isotypes have been identified which display a distinct pattern of tissue expression. Previously we found that $PLC-{\gamma}\;1$ and one of four ${\beta}-tubulin$ isotypes including ${\beta}1$, ${\beta}2$, ${\beta}3$ and ${\beta}6$, colocalized in COS-7 cells and cotranslocated to the plasma membrane to activate $PLC-{\gamma}\;1$ upon agonist stimulation. In the present study, we demonstrate that the remaining two, tubulin ${\beta}4$ and ${\beta}5$, also showed a potential to activate $PLC-{\gamma}\;1$. The phosphatidylinositol 4,5-bisphosphate $(PIP_2)$ hydrolyzing activity of $PLC-{\gamma}\;1$ was substantially increased in the presence of purified ${\beta}4$ and ${\beta}5$ tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that tubulin ${\beta}4$ and ${\beta}5$ also activate $PLC-{\gamma}\;1$. Taken together, our results suggest that all the ${\beta}-tubulin$ isotype activates $PLC-{\gamma}\;1$ activity to regulate cellular signaling. 포스포리파제 C-감마1$(phospholipase\;C-{\gamma}\;1;\;PLC-{\gamma}\;1)$은 활성화될 경우 세포 내의 이차전령인 inositol 1,4,5-trisphosphate$(IP_3)$와 diacylglycerol(DG)을 생성하는 중요한 세포 신호전달 분자이다. 튜불린은 미세소관과 방추사의 주요 구성 단백질로서 알파형과 베타형의 두 가지 동위형이 있는데 이들은 모든 진핵세포에 존재하면서 이형 이합체를 형성한다. 이 중 베타형 튜불린은 사람의 경우 6종의 또 다른 동위형이 존재하는 것으로 밝혀졌는데 이들은 각 조직에서 그 발현양상이 서로 다르게 나타난다. 이전의 연구에서 우리들은 $PLC-{\gamma}\;1$과 4종의 베타튜불린 동위형 즉, ${\beta}1$, ${\beta}2$, ${\beta}3$ 및 ${\beta}6$이 세포 내에서 서로 결합할 수 있으며 또한 외부의 자극이 전달될 경우 이들 4종의 동위형이 $PLC-{\gamma}\;1$을 활성화시켜 준다는 사실을 보고한 바 있다. 이번 실험에서는 이전의 연구에서 조사하지 못하였던 베타 튜불린의 나머지 두 가지 동위형 즉, ${\beta}4$및 ${\beta}5$가 $PLC-{\gamma}\;1$에 결합하여 $PLC-{\gamma}\;1$의 활성을 증가시켜줌으로서 세포 내에서의 신호전달계를 조절하고 있음을 확인하였다. 이 결과는 이전의 연구결과와 연관 지워볼 때, 6종의 모든 베타형 튜불린은 세포 외부의 자극이 있을 경우 $PLC-{\gamma}\;1$을 활성화시켜줌을 시사한다.

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.

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.

Molecular Events of Insulin Action Occur at Lipid Raft/Caveolae in Adipocytes

배순식,윤성지,김은경,김치대,최장현,서판길,Bae, Sun-Sik,Yun, Sung-Ji,Kim, Eun-Kyung,Kim, Chi-Dae,Choi, Jang-Hyun,Suh, Pann-Ghill Korean Society of Life Science 2007 생명과학회지 Vol.17 No.1

인슐린은 지방세포 또는 근육세포에서 포도당 흡수 조절 통로단백질이 함유되어 있는 소포제를 세포막으로의 이동을 촉진시킨다. 우리는 여기서 지방세포로의 분화는 인슐린에 의한 포도당 흡수에 대한 반응이 증가됨을 보였다. 반면에 지방세포로의 분화는 PDGF에 의한 포도당 흡수 반응이 감소됨을 보였다. 인슐린 수용체나 caveolae는 지방세포로의 분화과정 동안 발현이 증가된다. 또한 지방세포로의 분화는 인슐린에 의한 Akt의 활성을 증가시켰다. 하지만 PDGF에 의한 Akt의 활성은 크게 감소하였다. 하지만 인슐린은 지방세포 또는 섬유아 전구세포에서 ERK의 활성을 유도하지 않았다. PDGF에 의한 ERK 활성 또한 지방세포로의 분화과정에 따라 감소하였다. P13K의 저해제인 LY294002는 지방세포 뿐만 아니라 섬유아 전구세포에서 인슐린에 의한 포도당 흡수를 저해하였다. 마지막으로 인슐린 수용체, Akt, SHIP2, p85등이 lipid raft/caveolae에 존재함을 확인하였고 인슐린에 의해 이런 단백질들이 lipid raft/caveolae로 이동함을 관찰하였다. 이런 결과를 토대로 lipid raft는 포도당 홉수를 위한 인슐린의 기능적 작용을 하는데 매우 중요한 환경을 제공함을 주장한다. Insulin stimulates the fusion of intracellular vesicles containing glucose transporter 4 (GLUT4) with plasma membrane in adipocytes and muscle cells. Here we show that adipocyte differentiation results in enhanced insulin sensitivity of glucose uptake. On the other hand, glucose uptake in response to platelet-derived growth factor (PDGF) stimulation was markedly reduced by adipocyte differentiation. Expression level of insulin receptor and caveolin-1 was dramatically increased during adipocyte differentiation. Adipocyte differentiation caused :ilightly enhanced activation of acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) by insulin stimulation. However, activation of Akt by PDGF stimulation was largely reduced. Activation of ERK was not detected in both fibroblasts and adipocytes after stimulation with insulin. PDGF-dependent activation of ERK was reduced by adipocyte differentiation. Insulin-dependent glucose uptake was abrogated by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, in both fibroblasts and adipocytes. Also disassembly of caveolae structure by $methyl-\beta-cyclodextrin$ caused impairment of Akt activation and glucose uptake. Finally, insulin receptor, Akt, SH2-domain-containing inositol 5-phosphatase 2 (SHIP2), and regulatory subunit of PI3K are localized at lipid raft domain and the translocation was facilitated upon insulin stimulation. Given these results, we suggest that lipid raft provide proper site for insulin action for glucose uptake.

Direct tyrosine phosphorylation of Akt/PKB by epidermal growth factor receptor

배순식,최장현,윤성지,김은경,오용석,김치대,서판길,Bae, Sun-Sik,Choi, Jang-Hyun,Yun, Sung-Ji,Kim, Eun-Kyung,Oh, Yong-Suk,Kim, Chi-Dae,Suh, Pann-Ghill Korean Society of Life Science 2007 생명과학회지 Vol.17 No.2

Akt/PKB는 세포의 증식, 분화, 사멸, 혈관신생 등 매우 많은 생리활성 조절에 있어 매우 중요한 역할을 수행한다. 우리는 Akt/PKB의 tyrosine잔기의 인산화가 $Thr^{\308}$ 인산화에 필수적임을 밝혔다. COS-7 세포주에 EGF를 처 리하면 Akt/PKB의 tyrosine 잔기에 인산화가 촉진되었으며 이러한 인산화 촉진은 Akt/PKB에 myristoylation site를 이용해 세포막으로 이동시키면 더욱 더 증가하였다. 특히, 분리된 Akt/PKB와 EGF 수용체를 이용해 인산화 반응을 실시하면 tyrosine잔기의 인산화뿐만 아니라 $Ser^{\473}$에 대한 인산화도 증가하였다. 더욱이 tyrosine잔기에 인산화 된 Akt/PKB는 활성화된 EGF 수용체와 직접적인 결합을 이루고 있음을 확인하였다. 마지막으로 예측되는 tyrosine 잔기인 $(Tyr^{\326})$을 Alanine으로 치환하면 정상 Akt/PKB뿐만 아니라 활성화된 Akt/PKB의 EGF에 의한 $Thr^{\308}$ 인산화가 사라짐을 확인하였다. 이러한 결과들을 바탕으로 EGF 수용체에 의한 직접적인 Akt/PKB의 tyrosine 인산화는 EGF에 의한 많은 생리활성 조절기전의 또 다른 기전이라 볼 수 있다. Akt/PKB plays pivotal roles in many physiological responses such as proliferation, differentiation, apoptosis, and angiogenesis. Here we show that tyrosine phosphorylation of Akt/PKB is essential for the subsequent phosphorylation at $Thr^{\308}$. Tyrosine phosphorylation of Akt/PKB was induced by stimulation of COS-7 cells with epidermal growth factor receptor (EGF) and its phosphorylation was significantly enhanced by constitutive targeting of Akt/PKB to the plasma membrane by myristoylation. Interestingly, incubation of affinity purified Myc-tagged Akt/PKB with purified EGF receptor resulted in tyrosine phosphorylation as well as $Ser^{\473}$ phosphorylation of Akt/PKB. In addition, tyrosine-phosphorylated Akt/PKB could directly associate with activated EGF receptor in vitro. Finally, alanine mutation at putative tyrosine phosphorylation site $(Tyr^{\326})$ abolished EGF induced $Thr^{\308}$ phosphorylation of wild type as well as constitutively active form of Akt/PKB. Given these results we suggest here that direct tyrosine phosphorylation of Akt/PKB by EGF receptor could be another mechanism of EGF-induced control of many physiological responses.

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.

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.

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.