ErbB2 kinase domain is required for ErbB2 association with β-catenin

하남출,슈완핑,넥컬즈렌,정연진,Ha, Nam-Chul,Xu, Wanping,Neckers, Len,Jung, Yun-Jin Korean Society of Life Science 2007 생명과학회지 Vol.17 No.3

To investigate the region of ErbB2 for the $ErbB2-{\beta}-catenin$ interaction, a proteasome $resistant-{\beta}-catenin$ and various ErbB2 constructs were transfected in COS7 cells. ErbB2 proteins were immunoprecipitated, and coimmunoprecipitated ${\beta}-catenin$ was examined by Western blotting. ${\beta}-catenin$ coimmunoprecipitated with full length ErbB2. Of the truncated ErbB2 proteins DT (1-1123), DHC (1-1031) and DK (1-750), the ErbB2 constructs containing the kinase domain, DT and DHC, precipitated together with ${\beta}-catenin$ but DK containing no kinase domain did not. To further test the requirement of the kinase domain for ${\beta}-catenin-ErbB2$ interaction, the presence of ${\beta}-catenin$ in the immunocomplex was examined following transfection with an ErbB2 mutant (${\triangle}750-971$) whose kinase domain is internally deleted and subsequent immunoprecipitation of the ErbB2 mutant. ${\beta}-catenin$ was not detected in the immunocomplex. These results suggest that the ErbB2 kinase domain comprises a potential site for ${\beta}-catenin$ binding to the receptor tyrosine kinase. ${\beta}-catenin$과 결합하는 ErbB2의 부위를 조사하기 위하여 proteasome에 의하여 분해되지 않는 ${\beta}-catenin$과 다양한 ErbB2 construct를 COS7 세포에 transfection한 후 ErbB2 단백질을 그것의 항체로 가라앉혔다. 이 때 공침한 ${\beta}-catenin$을 Western blot으로 분석하였다. C 말단에서부터 잘려진 ErbB2 단백질 중에 kinase 영역을 가지고 있는 것들만 ${\beta}-catenin$과 공침하였다. kinase 영역의 필요성을 확인하기 위하여 kinase 영역이 내부에서 제거된 ErbB2 construct를 ${\beta}-catenin$과 transfection 한 후 동일한 실험을 실시하였다. 이 실험에서 ${\beta}-catenin$는 kinase 영역이 내부적으로 제거된 ErbB2 단백질과 공침하지 않았다. 이 결과는 ${\beta}-catenin$과 결합하는 ErbB2의 위치는 kinase 영역내에 있음을 제시한다.

Puromycin aminonucleoside 투여에 따른 사구체 족세포 ${\beta}$-catenin의 변화

최지영,안은미,박혜영,신재일,하태선,Choi, Ji-Young,Ahn, Eun-Mi,Park, Hye-Young,Shin, Jae-Il,Ha, Tae-Sun 대한소아신장학회 2011 Childhood kidney diseases Vol.15 No.2

목적 : 단백뇨 질환의 실험모델인 puromycin aminonucleoside (PAN)에서 관찰할 수 있는 족세포의 병리학적 이상에 있어서 ${\beta}$-catenin의 변화를 생체 외 족세포 배양실험을 통하여 알아보고자 하였다. 방법: PAN에 의한 족세포의 ${\beta}$-catenin의 변화를 생체 외 배양실험을 통해 알아보고자 백서 사구체 상피세포를 배양하여 다양한 농도의 PAN을 투여하여 confocal 현미경을 통하여 ${\beta}$-catenin의 분포를 관찰하였고, Western blotting과 RT-PCR을 사용하여 ${\beta}$-catenin 발현의 변화를 관찰하였다. 결과:외곽세포질에 분포하는 ${\beta}$-catenin이 단일세포 혹은 응집환경에서 PAN의 농도가 올라갈수록 흐려지면서 세포간에 간극이 생기는 것을 볼 수 있었다. Western 분석에서, ${\beta}$-catenin 단백양은 PAN의 농도가 증가할수록, 특히, 고농도인 $50{\mu}g/mL$에서 24시간과 48시간이 노출 조건에서 각각 34.9%와 34.3%의 의미 있는 감소소견을 보였다(P<0.05). 이러한 소견은 RT-PCR에서도 유사하게 보였으며, 24시간에서는 고농도인$50{\mu}g/mL$ PAN을 첨가한 조건에서 25.4%의 의미 있는 감소를 보였으며, 48시간에서는 $25{\mu}g/mL$와 $50{\mu}g/mL$ PAN을 첨가한 조건에서 각각 46.6%와 51.8%의 의미 있는 감소를 보였다. 결론: PAN은 족세포에서 ${\beta}$-catenin을 세포막으로부터 내부로의 분포변화를 유발하고, ${\beta}$-cate-nin mRNA의 발현 감소와 단백수준에서 양의 감소를 초래함으로서, PAN에 의한 족세포 내 분포변화에 유전자 억제에 의한 ${\beta}$-catenin 단백의 감소로 단백뇨의 발생에 기여할 것이라 사료된다. Purpose : To test whether the expression of ${\beta}$-catenin, a component of podocyte as a filtration molecule, would be altered by puromycin aminonucleoside (PAN) in the cultured podocyte in vitro. Methods : We cultured rat glomerular epithelial cells (GEpC) with various concentrations of PAN and examined the distribution of ${\beta}$-catenin by confocal microscope and measured the change of ${\beta}$-catenin expression by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Results :We found that ${\beta}$-catenin relocalized from peripheral cytoplasm to inner cytoplasm, therefore, intercellular separations were seen in confluently cultured cells by high concentrations of PAN in immunofluorescence views. In Western blotting of GEpC, PAN ($50{\mu}g/mL$) decreased ${\beta}$-catenin expression by 34.9% at 24 hrs and 34.3% at 48 hrs, compared to those in without PAN condition (P<0.05). In RT-PCR, high concentrations ($50{\mu}g/mL$) of PAN also decreased ${\beta}$-catenin mRNA expression similar to protein suppression by 25.4% at 24 hrs and 51.8% at 48 hrs (P<0.05). Conclusion : Exposure of podocytes to PAN in vitro relocates ${\beta}$-catenin internally and reduces ${\beta}$-catenin mRNA and protein expression, which could explain the development of proteinuria in experimental PAN-induced nephropathy.

Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the expression of ${\beta}$-catenin, leading to a rapid proliferation of pancreatic cells

Cho, Il-Rae,Koh, Sang-Seok,Min, Hye-Jin,Kim, Su-Jin,Lee, Yang-Soon,Park, Eun-Hee,Ratakorn, Srisuttee,Jhun, Byung-Hak,Oh, Sang-Taek,Johnston, Randal N.,Chung, Young-Hwa Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.2

It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of ${\beta}$-catenin while the suppression of PAUF by shRNA down-regulates ${\beta}$-catenin. The induction of ${\beta}$-catenin by PAUF is mediated by the activities of Akt and GSK-$3{\beta}$, but inhibition of downstream ERK does not reduce ${\beta}$-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of ${\beta}$-catenin, we examined the phosphorylation status of ${\beta}$-catenin in the presence of PAUF compared with that of ${\beta}$-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of ${\beta}$-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of ${\beta}$-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both $cyclin-D1$ and $c-Jun$, target genes of ${\beta}$-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of ${\beta}$-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize ${\beta}$-catenin $via$ a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.

참그물 바탕말 추출물에 의한 Wnt/${\beta}$-Catenin 신호전달체계 저해

조문주,오상택,Cho, Munju,Oh, Sangtaek 한국해양바이오학회 2006 한국해양바이오학회지 Vol.1 No.4

Wnt/${\beta}$-catenin 신호전달계의 비정상적인 활성화는 ${\beta}$-catenin response transcription (CRT)를 증가시킬 뿐 아니라 대장암의 발생과 밀접한 관련이 있다. 따라서 Wnt/${\beta}$-catenin 신호전달계는 대장암에 대한 항암제 개발 및 대장암 치료에 중요한 표적이 된다. 본 연구에서는 세포기반 초고속 스크리닝 기법을 사용하여 Wnt/${\beta}$-catenin 신호를 저해하는 참그물 바탕말 추출물을 발굴하였다. 참그물 바탕말 추출물은 세포내 ${\beta}$-catenin 단백질 수준에는 영향을 미치지 않았으며 ${\beta}$-catenin/TCF에 의해 조절되는 유전자 중의 하나인 cyclin D1의 발현을 저해하였다. 또한 참그물 바탕말 추출물은 다양한 대장암의 증식을 저해하였다. 본 연구의 결과들로부터 참그물 바탕말 추출물이 잠재적으로 대장암을 포함하는 새로운 암 예방 및 치료제로 사용될 수 있을 것이다. Abnormal activation of the Wnt/${\beta}$-catenin pathway and subsequent up-regulation of ${\beta}$-catenin response transcription (CRT) are associated with the development of colon cancer. Thus, the Wnt/${\beta}$-catenin pathway is an attractive target for chemoprevention and treatment of this cancer. In this study, we used a cell-based screen to identify a methanol extract of Dictyota dichotoma (EDD) that suppresses the Wnt/${\beta}$-catenin pathway without altering the level of ${\beta}$-catenin protein and reduces the expression of cyclin D1, which is a known ${\beta}$-catenin/T cell factor (TCF)-dependent gene. EDD inhibited the growth of various colon cancer cells. Our findings suggest that EDD can potentially be used as a chemopreventive agent against colon cancer.

조기 위암에서 Beta-catenin과 E-cadherin의 발현 분석

송병주 ( Song Byeong Ju ),박영진 ( Park Yeong Jin ),김한성 ( Kim Han Seong ),김철남 ( Kim Cheol Nam ),장석효 ( Jang Seog Hyo ) 대한소화기학회 2004 대한소화기학회지 Vol.43 No.2

Background/Aims: Beta-catenin is known to perform two unrelated functions in cadherin-mediated cell to cell adhesion system and Wnt pathway. Recent studies reported cytoplasmic and nuclear accumulation of beta-catenin by Wnt signaling and/or abnormal Wnt pathway in cancer cells. Nuclear accumulations of beta-catenin have a crucial role in early tumor growth and initiation of invasive growth in gastric cancer. Methods: We carried out clinicopathological and immunohistochemical studies for beta-catenin, p53, E-cadherin, and Ki-67 in the specimens from 60 early gastric cancer patients who were treated with curative resections. Results: Twenty-five (41.7%) and twenty-nine (48.3%) cases showed a nuclear and cytoplasmic expression of beta-catenin, respectively. There were significant correlations between nuclear expression of beta-catenin and well-differentiated and intestinal type of early gastric carcinoma. Cytoplasmic expression of beta-catenin had significant correlations with nuclear expression of beta-catenin (p=0.011). Conclusions: Nuclear expression of beta-catenin is significantly influenced by histological grade, Lauren classification and cytoplasmic expression of beta-catenin in early gastric cancer. These findings suggest that nuclear expression of beta-catenin is correlated with early tumorigenesis and initiation of invasive growth in gastric cancer. (Korean J Gastroenterol 2004;43: 82-89)

Silybin Synergizes with Wnt3a in Activation of the Wnt/${\beta}$-catenin Signaling Pathway through Stabilization of Intracellular ${\beta}$-Catenin Protein

김태연,오상택,Kim, Tae-Yeoun,Oh, Sang-Taek The Korean Society for Microbiology and Biotechnol 2012 한국미생물·생명공학회지 Vol.40 No.1

Wnt/${\beta}$-catenin 신호전달체계는 세포의 분화와 증식, 기관의 발생과 조절을 담당하는 중요한 세포내 신호전달체계이다. 발생과정에서 Wnt/${\beta}$-catenin 신호전달체계의 작용이 지방세포로의 분화를 억제하고 조골세포와 신경세포로의 분화는 촉진한다는 많은 연구들이 보고되어 있으며, 현재 Wnt/${\beta}$-catenin 신호전달체계의 조절을 통한 여러 질병의 치료와 예방에 대한 관심이 대두되고 있다. 본 연구에서는 세포를 기반으로 한 초고속 저분자 스크리닝 시스템을 이용하여 Wnt의 상승제인 silybin을 발굴하였다. silybin은 Wnt가 존재 않을 경우에는 ${\beta}$-catenin 단백질의 수준에 영향을 미치지 않지만 Wnt가 존재할 경우, mRNA 발현양의 변화 없이 세포질내의 ${\beta}$-catenin 단백질의 수준을 증가시킨다. 또한 silybin에 의해 증가된 ${\beta}$-catenin으로 인해 지방세포분화에 중요한 전사인자라고 알려진 PPAR-${\gamma}$와 C/EBP-${\alpha}$의 발현을 억제한다. 따라서 이 연구에서는 silybin이 세포질내 ${\beta}$-catenin 단백질의 수준을 증가시킴으로써 Wnt/${\beta}$-catenin 신호전달체계를 활성한다는 사실을 제시하였다. The Wnt/${\beta}$-catenin signaling pathway regulates diverse developmental processes and adult tissue homeostasis. Inappropriate regulation of this pathway has been associated with human diseases, such as cancers, osteoporosis, and Alzheimer's disease. Using a cell-based chemical screening with natural compounds, we discovered silybin, a plant flavonoid isolated from the Silybum marianum, which activated the Wnt/${\beta}$-catenin signaling pathway in a synergy with Wnt3a-conditioned medium (Wnt3a-CM). In the presence of Wnt3a-CM, silybin up-regulated ${\beta}$-catenin response transcription (CRT) in HEK293-FL reporter cells and 3T3-L1 preadipocytes through stabilization of intracellular ${\beta}$-catenin protein. Silybin and Wnt3a-CM synergistically reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated $receptor{\gamma}$ ($PPAR{\gamma}$) and CAATT enhancer-binding protein ${\alpha}$ (C/$EBP{\alpha}$) in 3T3-L1 preadipocytes, accompanied by the activation of Wnt/${\beta}$-catenin signaling pathway. Taken together, our findings indicate that silybin is a small-molecule synergist of the Wnt/${\beta}$-catenin signaling pathway and can be used as a controllable reagent for investigating biological processes that involve the Wnt/${\beta}$-catenin signaling pathway.

장형 위암에서, pS2, c-MYC, beta-catenin, APC 단백을 포함한 APC유전자경로의 역할

Chang, Heekyung KOSIN UNIVERSITY COLLEGE OF MEDICINE 2006 高神大學校 醫學部 論文集 Vol.21 No.1

배경 : 1998년 가족성대장용종증 환자의 비종양성 상피세포에서 APC유전자가 beta-catenin/Tcf-4 유전자를 통한 c-MYC 유전자를 직접 조정한다는 가설이 보고되었다. pS2가 속하는 intestinal trefoil factor(TTF)가 beta-catenin가 관련되는 위-특이 종양억제 유전자일 것일라는 보고도 있었다. 따라서 위암발생에 있어서도 대장암 발생과 같이 beta-catenin, c-MYC과 관련된 APC 경로가 적용될 수 있는 지 또, pS2를 이 경로에 포함시킬 수 있는지를 조사하고자 하였다. 대상 및 방법 : 대장암과 형태학적으로 유사한 장형 위암을 30례의 포르말린에 고정되고 파라핀에 포매된 위암조직과 주위 조직을 대상으로 면역 염색을 실시하였다. 결과 : pS2의 면역 염색결과는 56.7%, c-MYC는 43.3%, beta-catenin은 86.7%의 양성율은 나태내었다. 특히, beta-catenin은 종양의 침윤 상태에 따라 다양한 면역 염색 반응을 보였다. 종양이 침윤하는 가장 깊은 부분인 최전면부에서는 강한 양성 반응을 보이는 반면, 그위의 점막에 있는 종양에서는 발현되지 않았다. 결론 : 위암중 장형암은 대장암에서와 같은 APC 경로의 이상변형으로 암이 발생한다는 가설은 아직은 과학적 근거는 부복한 것으로 생각되나, beta-catenin은 세포질의 단백이 핵내로 이동하여 암화 과정에 관여하는 것과 동시에 종양이 침윤하는 최전면부에서는 세포간 부착 단백으로서의 성질을 소실하여 종양이 세포외 기질로 침윤하도록 한다고 생각된다. Background: A proposal that in normal colorectal epithelial cells, the adenomatous polyposis coli gene (APC) might modulate directly c-MYC transcription through beta-catenin/Tcf-4 was reported in 1998. It was reported that intestinal trefoil factor (TTF) which is the same family of pS as a gastric - specific tumor suppressor gene is related to beta-catenin. Method and Material: To elucidate the applicability of APC pathway in stomach and relationship between this pathway and pS2, the expressions of pS2, c-MYC, and beta-catenin proteins wereevaluated by immunohistochemically in 30 cases of intestinal carcinoma of stomach which is morphologically close to colon cancer. Restults: Their immunoreactivities were 56.7% for pS2, 43.3% for c-MYC, and 86.7% for beta-catenin proteins, respectively. Interestingly, positive staining of beta-catenin showed heterogenous pattern according to the depth of invasion. Tumor cells in the invading or infiltrating edges showed strong positivity, while thetumor cells in mucosa showed loss of the expression even ib the same tumors. Conclusion: This results suggest that in intestinal carcinoma of stomach, beta-catenin involves in the tumor celll migration and invasion not as cell adhesion molecule but as tumor-suppressor molecule forming complex with APC. In addition, the expressions of c-MYC and pS2 was not correlated with the function of adhesion molecule of beta-catenin

STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells

Shin, Minkyung,Yi, Eun Hee,Kim, Byung-Hak,Shin, Jae-Cheon,Park, Jung Youl,Cho, Chung-Hyun,Park, Jong-Wan,Choi, Kang-Yell,Ye, Sang-Kyu Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.11

The ${\beta}$-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, ${\beta}$-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of ${\beta}$-catenin. The level of ${\beta}$-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to ${\beta}$-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and ${\beta}$-catenin in HEK293T cells. To our knowledge, this is the first study to report that ${\beta}$-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated ${\beta}$-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active ${\beta}$-catenin via degradation, which stabilized SIAH-1 and increased its interaction with ${\beta}$-catenin. These results suggest that activated STAT3 regulates active ${\beta}$-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of ${\beta}$-catenin in HEK293T cells.

2-Deoxy-D-glucose regulates dedifferentiation through ${\beta}$-catenin pathway in rabbit articular chondrocytes

Yu, Seon-Mi,Kim, Hyun-Ah,Kim, Song-Ja Korean Society for Biochemistry and Molecular Bion 2010 Experimental and molecular medicine Vol.42 No.7

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the ${\beta}$-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and ${\beta}$-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of ${\beta}$-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of ${\beta}$-catenin, as the 2DG stimulated accumulation of ${\beta}$-catenin, which is characterized by translocation of ${\beta}$-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of ${\beta}$-catenin degradation by inhibition of glycogen synthase kinase 3-${\beta}$ with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of ${\beta}$-catenin whereas the transcriptional level of ${\beta}$-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via ${\beta}$-catenin pathway in rabbit articular chondrocytes.

표피종양에서 Syndecan-1, E-cadherin, Beta-catenin 발현에 관한 면역조직화학적 연구

신봉석 ( Bong Seok Shin ),최규철 ( Kyu Chul Choi ),김해련 ( Hae Ryun Kim ),김민성 ( Min Sung Kim ) 대한피부과학회 2011 大韓皮膚科學會誌 Vol.49 No.4

Background: Syndecan-1, E-cadherin and beta-catenin are cell adhesion molecules that are primarily expressed on the surface of adult epithelial cells. The expressions of them appear to be inversely correlated with tumor aggressiveness and invasiveness. Objective: The purpose of this study was to investigate the expressions of syndecan-1, E-cadherin and beta-catenin in tissue sections of normal sun-damaged skin, cutaneous premalignant lesions and squamous cell carcinoma in all stages of the evolution of lesions associated with sun exposure. Methods: Ten normal skins from patients with actinic keratosis, 10 cases of seborrheic keratosis, 5 cases of actinic keratosis, 5 cases of Bowen`s disease, 5 cases of keratoacanthoma without dysplatic cells, 5 cases of keratoacanthoma with dysplastic cells in an invasive margin, 5 cases of poor-differentiated squqmous cell carcinoma and 5 cases of acantholytic squamous cell carcinoma were investigated. The specimens were assessed for the syndecan-1, E-cadherin and beta-catenin expressions using a semi-quantitative method in which the intensity of membranous staining was evaluated. Results: In almost all the cases the expression of the E-cadherin and beta-catenin was very similar. These adhesion molecules were progressively reduced in the epidermis of normal sun-damaged skin through premalignant lesions to squamous cell carcinoma. Also, the expression of syndecan-1 was similar to the E-cadherin and beta-catenin expressions except for a normal expression in premalignant lesions. But all three adhesion molecules were diminished with decreasing cell differentiation. Conclusion: Our results suggest that syndecan-1, E-cadherin and beta-catenin are expressed similarly in the epithelium, and that the decreased expression of these adhesion molecules is associated with malignant transformation. (Korean J Dermatol 2011;49(4):309∼317)