• 생명과학회지
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• 제32권2호
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• pp.148-154
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• 2022

국소접착인산화효소(FAK)는 국소접착부에서 세포부착, 세포이동, 세포역학적 신호전달 등에 관여한다고 알려져 있다. 그러나 세포 외 기질(ECM)과 상호작용하는 인테그린 막단백질과 함께 위치하는 세포막 미세영역(membrane microdomain)의 종류와 ECM 구성에 따른 FAK 활성은 여전히 불분명하다. 형광 공명 에너지 전달(FRET)을 기반으로 유전적으로 인코딩 된 바이오센서는 세포 내 FAK 신호를 높은 시공간 해상도로 제공할 수 있다. 본 연구에서는 유리, 제1형 콜라겐, 피브로넥틴, 라미닌의 ECM 조건에서 FRET 기반 막 표적 FAK 바이오센서를 사용하여 지질유동섬(Lipid raft) 및 비-지질유동섬(non-Lipid raft)에서 FAK의 활성을 분석하고 시각화 하였다. 흥미롭게도, 지질유동섬에서 라미닌 조건 하의 FAK 활성은 다른 ECM 조건보다 낮았고, 비-지질유동섬에서 FAK 활성은 다른 ECM 조건보다 낮았다. 동일한 ECM 조건 상의 비교에서는 피브로넥틴 조건일 때 지질유동섬에서 비-지질유동섬 보다 높은 FAK 활성이 관측되었다. 따라서 이번 연구는 FAK 활성도가 ECM 유형 및 세포막 미세영역에 따라 특이적으로 조절되는 것을 시각적, 정량적으로 보여준다.

• BMB Reports
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• 제54권2호
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• pp.112-117
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• 2021

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK.

• 대한의용생체공학회:의공학회지
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• 제40권1호
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• pp.1-6
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• 2019

Human mesenchymal stem cells (hMSC) are multipotent stromal cells that have great potential to differentiate into a variety of cell types such as osteocytes, chondrocytes, and myocytes. Although there have been many studies on their clinical availability, little is known about how intracellular signals can be modulated by topographic features of the extracellular matrix (ECM). In this study, we investigated whether and how microwavy-patterned extracellular matrix (ECM) could affect the signaling activity of focal adhesion kinase (FAK), a key cellular adhesion protein. The fluorescence resonance energy transfer (FRET)-based FAK biosensor-transfected cells are incubated on microwavy-patterned surfaces and then platelet derived growth factor (PDGF) are treated to trigger FAK signals, followed by monitoring through live-cell FRET imaging in real time. As a result, we report that PDGF-induced FAK was highly activated in cells cultured on microwavy-patterned surface with L or M type, while inhibited by H type-patterned surface. In further studies, PDGF-induced FAK signals are regulated by functional support of actin filaments, microtubules, myosin-related proteins, suggesting that PDGF-induced FAK signals in hMSC upon microwavy surfaces are dependent on cytoskeleton (CSK)-actomyosin networks. Thus, our findings not only provide new insight on molecular mechanisms on how FAK signals can be regulated by distinct topographical cues of the ECM, but also may offer advantages in potential applications for regenerative medicine and tissue engineering.

• 통합자연과학논문집
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• 제4권4호
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• pp.266-272
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• 2011

Focal adhesion kinase (FAK) is a potential target for the treatment of primary cancers as well as prevention of tumor metastasis. To understand the structural and chemical features of FAK inhibitors, we report comparative molecular field analysis (CoMFA) for the series of 7H-pyrrolo(2,3-d)pyrimidines. The CoMFA models showed good correlation between the actual and predicted values for training set molecules. Our results indicated the ligand-based alignment has produced better statistical results for CoMFA ($q^2$ = 0.505, $r^2$ = 0.950). Both models were validated using test set compounds, and gave good predictive values of 0.537. The statistical parameters from the generated 3D-QSAR models were indicated that the data are well fitted and have high predictive ability. The contour map from 3D-QSAR models explains nicely the structure-activity relationships of FAK inhibitors and our results would give proper guidelines to further enhance the activity of novel inhibitors.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2897-2901
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• 2012

Invasion is usually recognized as the main reason for the high recurrence and death rates of glioma and restricts the efficacy of surgery and other therapies. Therefore, we aimed to investigate the mechanism involved in promotion effects of mda-9/syntenin on human glioma cell migration. The wound healing method was used to test the migration ability of human glioma cells CHG-5 and CHG-hS, stably overexpressing mda-9/syntenin. Western blotting was performed to determine the expression and phosphorylation of focal adhesion kinase (FAK) and JNK in CHG-5 and CHG-hS cells. The migration ability of CHG-hS cells was significantly higher than that of CHG-5 cells in fibronectin (FN)-coated culture plates. Phosphorylation of FAK on tyrosine 397, 576, and 925 sites was increased with time elapsed in CHG-hS cells. However, phosphorylated FAK on the tyrosine 861 site was not changed. Phosphorylated Src, JNK and Akt levels in CHG-hS cells were also significantly upregulated. Phosphorylation of JNK and Akt were abolished by the specific inhibitors SP600125 and LY294002, respectively, and the migration ability of CHG-hS cells was decreased, indicating that the JNK and PI3K/Akt pathways play important roles in regulating mda-9/syntenin-induced human brain glioma migration. Our results indicate Mda-9/syntenin overexpression could activate FAK-JNK and FAK-Akt signaling and then enhance the migration capacity of human brain glioma cells.

• Molecules and Cells
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• 제25권2호
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• pp.184-195
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• 2008

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP ($c-FLIP_L$), but not the short form ($c-FLIP_S$), enhanced adhesion and motility. Downregulation of $c-FLIP_L$ with siRNA decreased phosphorylation of FAK and ERK, while overexpression of $c-FLIP_L$ increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed $c-FLIP_L$-promoted motility. Inhibition of ROCK by Y27632 suppressed the $c-FLIP_L$-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of $c-FLIP_L$ increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced $c-FLIP_L$- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the $c-FLIP_L$-promoted cell motility. We conclude that $c-FLIP_L$ promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

• Clinical and Experimental Reproductive Medicine
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• 제33권2호
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• pp.115-123
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• 2006

목 적: 본 연구를 통해 GnRH 의한 세포 분열의 억제는 integrin, FAK 빛 ERK를 통한 세포 내 신호전달 기전을 통하여 일어남을 규명하고자 하였다. 연구방법: 연구에 사용된 인간자궁내막암 세포주는 DMEM/F12 (10% FBS)의 조건에서 배양 하였다. GnRH-I과 -II는 실험 목적에 따라 100 nM 농도로 0, 5, 10, 15, 20, 30분간 또는 10 nM or 100 nM의 농도로 20분간 처리 하였다. 세포의 분열 정도는 [$^3H$] thymidine incorporation assay를 이용하여 정량적으로 측정 하였으며, Immunoblotting 방법을 이용하여 단백질의 발현을 확인 하였다. 결 과: GnRH-I과 -II 모두 HEC1A 세포의 세보분열을 억제하였으며 integrin ${\beta}3$의 발현을 증가 시켰다. GnRH-I과 -II를 처리 후 FAK 및 ERK의 안산화가 증가됨을 관찰할 수 있었다. 결 론: GnRH에 의한 세포분열의 억제는 integrin의 발현과 FAK 및 ERK의 인산화 과정을 통하여 일어남을 알 수 있었다.

• 생명과학회지
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• 제22권1호
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• pp.16-24
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• 2012

Polyamine은 모든 세포의 성장과 분화에 필수적인 요소지만 그 기작은 아직 정확하게 밝혀져 있지 않다. 본 논문에서는 MCF-7세포에서 spm의 세포독성 기작을 연구하였다. MTT assay 결과 저농도의 spm (<10 ${\mu}M$) 처리시 cell viability가 증가하는 반면 고농도의 spm 처리시 처리 시간과 처리 농도에 의존적으로 감소되었으며, 이는 고농도의 spm이 MCF-7 cell에 cytotoxic한 효과를 가지고 있는 것으로 사료된다. Cell cycle 분석 결과 spm 농도에 의존적으로 sub-G1 단계의 세포 양이 증가하는 것으로 나타났으며, 이는 spm이 세포분열을 억제함으로써 세포사를 유발하는 것으로 생각된다. 또한 고농도의 spm 처리 후 2시간이 지나자 cell 표면이 움츠려 들며 rounding되기 시작하여 하루가 지난 후 거의 모든 cell이 culture dish 에서 떨어진 것을 관찰할 수 있었다. 이는 spm이 세포부착에 관여하여 세포사를 유발하는 것이라 생각되며, 세포부착을 조절하는 Integrin ${\beta}1$은 저농도의 spm에선 별다른 차이를 보이지 않다가 농도가 높아질수록 조금씩 감소하였으며, cytoskeletal protein인 actin은 농도의존적으로 감소하였다. 반면 adhesion protein인 FAK는 저농도의 spm 처리시에도 급격히 감소하였다. 이는 spm이 adhesion 및 cytoskeletal proteins의 발현을 억제하는 것으로 보이며, 특히 Integrin ${\beta}1$과 actin에 비해 FAK에 더 많은 영향을 끼치는 것으로 사료된다. 단백질들의 세포막상의 분포에 관한 연구에서, membrane 상에 위치하던 Integrin ${\beta}1$은 10 ${\mu}M$의 spm 처리시 세포 내로 약간의 위치변동이 일어났으나 그 양에는 크게 차이가 없었으며, 반면에 actin은 위치상엔 큰 변화가 일어나지는 않았지만 농도에 따라 크게 감소하는 것으로 나타났다. 세포이동과 형태조절에서 중추적인 역할을 하는 FAK는 세포막 안쪽에 위치하고 있다가 spm 처리시 세포 가운데로 이동하는 모습이 관찰되었으며 그 양도 크게 감소하였다. 이상의 실험에서 세포 내 spm의 변화는 MCF-7 cell의 adhesion protein인 Integrin ${\beta}1$과 FAK, 그리고 cytoskeletal protein인 actin의 발현을 조절하여 cell attachment를 억제함으로써 세포분열과 생장을 억제하는 것으로 분석된다.

• BMB Reports
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• 제57권6호
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• pp.305-310
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• 2024

T-plastin (PLST), a member of the actin-bundling protein family, plays crucial roles in cytoskeletal structure, regulation, and motility. Studies have shown that the plastin family is associated with the malignant characteristics of cancer, such as circulating tumor cells and metastasis, by inducing epithelial-mesenchymal transition (EMT) in various cancer cells. However, the role of PLST in the EMT of human lung cancer cells remains unclear. In this study, we observed that PLST overexpression enhanced cell migratory and invasive abilities, whereas its downregulation resulted in their suppression. Moreover, PLST expression levels were associated with the expression patterns of EMT markers, including E-cadherin, vimentin, and Slug. Furthermore, the phosphorylation levels of focal adhesion kinase (FAK) and AKT serine/threonine kinase (AKT) were dependent on PLST expression levels. These findings indicate that PLST induces the migration and invasion of human lung cancer cells by promoting Slug-mediated EMT via the FAK/AKT signaling pathway.

• International Journal of Oral Biology
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• 제37권4호
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• pp.189-195
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• 2012

Resistance to the induction of apoptosis is a possible mechanism by which tumor cells can survive anti-neoplastic treatments. Melanoma is notoriously resistant to anti-neoplastic therapy. Previous studies have demonstrated focal adhesion kinase (FAK) overexpression in melanoma cell lines. Given its probable role in mediating resistance to apoptosis, many researchers have sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting the PTK-mediated signaling pathway. This present study was undertaken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in the G361 melanoma cell line. Genistein was found to have a preferential cytotoxic effect on G361 melanoma cells over HaCaT normal keratinocytes. Genistein decreased the expression of 125 kDa phosphotyrosine kinase and the FAK protein in particular. Genistein treatment did not affect the expression of p53 in G361 cells in which p21 is upregulated. The expression of cyclin B and cdc2 was downregulated by genistein treatment. Taken together, our data indicate that genistein induces the decreased proliferation of G361 melanoma cells via the inhibition of FAK expression and regulation of cell cycle genes. This suggests that the use of genistein may be a viable approach to future melanoma treatments.