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protein tyrosine kinase and PI3-K participate in the inhibitory effect of insulin-like growth factor I on basolateral 10 pS Chloride channels in the thick ascending limb of the rat kidney

Protein tyrosine kinase and PI3-K participate in the inhibit...

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中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： Li-Jun WANG~1,Shu-Min KONG~1,Wen-Nan LI~1,Hong-Yu SUI~1,Peng WU~1,Ming-Xiao WANG~1,Yu-Jie LIU~1, Li-Li FAN~1,Li GU~1,Wen-Hui WANG~2,Rui-Min GU~(1,*) 1 Department of Pharmacology,Harbin Medical University,Harbin 150081,China 2 Department of Pharmacology,New York Medical College,Valhalla,New York,USA

＜正＞Insulin-like growth factorⅠ（IGF-I） plays an important role in accelerating protein and carbohydrate metabolism, and affects cell proliferation,differentiation and inhibiting apoptosis in a long *** to IGF-I and IGF-I receptor are distributed in the basolateral and apical membrane of the thick ascending limb,IGF-I could influence sodium chloride （NaCl） transport in this nephron *** a 10 pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment,thus we performed patch-clamp technique in cell-attached patches to study the effect of IGF-I on the basolateral 10 pS Cl channels in the thick ascending limb of the rat *** of IGF-I inhibited the basolateral 10 pS Cl channels,significantly decreased channel activity（a product of channel number and open probability）.The inhibitory effect of IGF-I is not mediated by protein tyrosine phosphatase or phospholipase C-dependent mechanisms,because blocking protein tyrosine phosphatase with phenylarsine oxide or inhibition of phospholipase C with U73122 did not abolish the inhibitory effect of IGF-I on the 10 pS Cl ***,inhibition of protein tyrosine kinase with Herbinmycin A or Genistein or repression of phosphatidylinositol 3-kinase（PI3-K） with Wortmannin abolished the effect of IGF-I,this suggests that protein tyrosine kinase and PI3-K are responsible for mediating the effect of IGF-I on the 10 pS Cl *** studies show that inhibition of basolateral 10 pS Cl channels by IGF-I is mediated by stimulating protein tyrosine kinase,which in turn stimulates PI3-K in the thick ascending limb of the rat kidney.

关键词： C1 transport insulin-like growth factor I protein tyrosine kinase phosphatidylinositol 3-kinase thick ascending limb

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Role of protein tyrosine kinase in IL-1β induced activation of mitogen- activated protein kinase in fibroblast-like synoviocytes of rheumatoid arthritis

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Chinese Medical Journal 2000年第10期000卷 8-12页

作者：吕厚山孙铁铮药立波张育军 Arthritis Clinic Research Center Departmen tof Gynecology Department of Biochemistry Forth Military Medical University

Obejctives To study mitogen activated protein kinase (MAPKs) activation in fibroblast like synoviocytes (FLS) of rheumatoid arthritis (RA) under the stimulation of IL 1β, and to elucidate the role of protein tyrosine kinase (PTK) in the activation of MAPKs Methods Primary cultures of RA FLS were used Western blot was applied to examine transient changes in protein tyrosine phosphorylation status and MAPKs activation in RA FLS stimulated with IL 1β at various doses, and over different periods Genistein, the specific PTK inhibitor, was used to evaluate the inhibitory role in activation of MAPKs by IL 1β Results IL 1β transiently increased protein tyrosine phosphorylation, and activated the MAPKs cascades (mainly ERK 2, JNK 2 and P 38 ) in RA FLS There was no obvious difference in MAPKs activation among different doses of IL 1β (1?IU/ml,10?IU/ml, 100?IU/ml), but the peak activation of ERK 2, JNK 2 and P 38 took place at 5?min, 15?min and 1?min, respectively, after stimulation with IL 1β The activation of ERK 2 was inhibited by genistein, but the inhibitory role on that of JNK and P 38 was relatively weak Conclusions During signal transduction of IL 1β in RA FLS, tyrosine phosphorylation was increased transiently, the MAPKs cascade was activated in a few minutes, and there was heterogenicity in the activation among three subfamily members PTK had a role in the activation of ERK, but had weak effects on that of JNK and P 38

关键词： arthritis fibroblasts IL 1 beta mitogen activated protein kinase protein tyrosine kinase genistein

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Csk-homologous kinase （Chk/Matk）： a molecular policeman suppressing cancer formation and progression

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Frontiers in Biology 2015年第3期10卷 195-202页

作者： Gahana Advani Anderly C. Chueh Ya Chee Lim Amardeep Dhillon Heung-Chin Cheng Department of Biochemistry and Molecular Biology Bio21 Molecular Science and Biotechnology Institute University of Melbourne 30 Flemington Road Parkville Victoria 3010 Australia A CRF Chemical Biology Division The Walter and Eliza Hall Institute for Medical Research Royal Melbourne Hospital and Department of Medical Biology University of Melbourne Parkville Victoria 3052 Australia Peter MacCallum Cancer Research Institute St. Andrews Place East Melbourne Hctoria Australia

Aberrant activation of Src-family tyrosine kinases （SFKs） directs initiation of metastasis and development of drug resistance in multiple solid tumors and hematological cancers. Since oncogenic mutations of SFKs are rare events, aberrant activation of SFKs in cancer is likely due to dysregulation of the two major upstream inhibitors： C-terminal Src kinase （Csk） and its homolog Csk-homologous kinase （Chk/Matk）. Csk and Chk/Matk inhibit SFKs by selectively phosphorylating the inhibitory tyrosine residue at their C-terminal tail. Additionally, Chk/Matk can also employ a non- catalytic inhibitory mechanism to inhibit multiple active forms of SFKs, suggesting that Chk/Matk is a versatile inhibitor capable of constraining the activity of multiple active forms of SFKs. Mounting evidence suggests that Chk/ Mark is a potential tumor suppressor downregulated by epigenetic silencing and/or missense mutations in several cancers such as colorectal and lung carcinoma. In spite of the potential significance of Chk/Matk in cancer, little is known about its structure and regulation. This review focuses on the mechanisms by which Chk/Matk expression and activity is downregulated in cancers. Specifically, we assessed the evidence demonstrating downregulation of Chk/Matk by epigenetic silencing and missense mutations in cancers. The other focus is the tumor suppressive mechanism of Chk/ Matk. The final focus of the review is on the clinical applications of the investigations into the mechanism of epigenetic silencing of Chk/Matk expression and the tumor suppressive mechanism of Chk/Matk; specifically we discussed how they can benefit the development of biomarkers for early diagnosis of cancers and specific SFK inhibitors for use as cancer therapeutics.

关键词： tumour suppressor protein tyrosine kinase Src-family kinases CSK CHK/Matk colon cancer

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kinases and glutathione transferases:selective and sensitive targeting

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Frontiers in Biology 2011年第2期6卷 156-169页

作者： Yasemin G.ISGOR Belgin S.ISGOR Chemistry Group Faculty of EngineeringAtilim UniversityAnkara 06836Turkey

kinases,representing almost 500 proteins in the human genome,are responsible for catalyzing the phosphorylation reaction of amino acid residues at their *** the largest family of kinases,the protein tyrosine kinases(PTKs)have roles in controlling the essential cellular activities,and their deregulation is generally related to pathologic *** recent efforts on identifying their signal transducer or mediator role in cellular signaling revealed the interaction of PTKs with numerous enzymes of different classes,such as Ser/Thr kinases(STKs),glutathione transferases(GSTs),and receptor tyrosine kinases(RTKs).In either regulation or enhancing the signaling,PTKs are determined in close interaction with these enzymes,under specific cellular conditions,such as oxidative stress and *** this concept,intensive research on thiol metabolizing enzymes recently showed their involvement in the physiologic functions in cellular signaling besides their well known traditional role in antioxidant *** shared signaling components between PTK and GST family enzymes will be discussed in depth in this research review to evaluate the results of recent studies important in drug targeting for therapeutic intervention,such as cell viability,migration,differentiation and proliferation.

关键词： glutathione transferase protein tyrosine kinase small molecule inhibitors c-Src signal transduction drug targeting

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Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2006年第1期7卷 13-19页

作者：龚兴国纪静谢捷周远张俊彦钟文涛 Institute of Biomacromolecule and Enzyme Engineering School of Life Sciences Zhejiang UniversityHangzhou 310027 China

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl β-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems em- ployed in earlier Src expression.

关键词： v-Src GST-fusion Inclusion body Orthogonalization protein tyrosine kinase

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NEW DRUG TARGETING TREATMENT-GLIVEC

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Chinese Journal of Cancer Research 2003年第3期15卷 235-239页

作者：孙雪梅 BRADY Ben Nanjing Drum Tower Hospital Nanjing University Medical College Nanjing 210008 China The Cabrini Medical Center Victoria 3050 Australia

This review evaluates the role of Glivec in the treatment of chronic myelogenous leukemia and other malignant tumors. Preclinical and clinical evidence showed that Glivec demonstrated a potent and specific inhibition on BCR-ABL positive leukemias and other malignant tumors in which overexpression of c-kit and PDGFR-b played a major role in their pathogenesis. Glivec has induced complete hematologic responses in up to 98% of patients evaluated in clinical trials. It's a very successful drug that supported the idea of targeted therapy through inhibition of tyrosine kinases. Although it's still in the early stages of clinical development and the resistance to Glivec remains to be a problem needed further study, a great deal has been learned from these research and observation. And with the increasing data, molecular targeting therapy will play much more important role in the treatment of malignant tumors. With the better understanding of the pathogenesis of malignant tumors, well-designed drugs targeting the specific molecular abnormalities with higher efficacy and lower side effect will benefit numerous patients with malignant tumors.

关键词： protein tyrosine kinase Glivec ST1571 Targeting therapy Hematopoietic malignancy BCR-ABL c-kit PDGFR-b

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Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells

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World Journal of Gastroenterology 2006年第3期12卷 420-425页

作者： Shi-Ying Li Bin-Bin Huang Shou Ouyang Xiamen Institute of Medicine Xiamen 361003 Fujian Province China

AIM： To investigate the action of genistein （GST）, a broad spectrum tyrosine kinase inhibitor, on voltagegated potassium channels in guinea pig proximal colon smooth muscle cells. METHODS： Smooth muscle cells in guinea pig proximal colon were enzymatically isolated. Nystatin-perforated whole cell patch clamp technique was used to record potassium currents including fast transient outward current （Ikto） and delayed rectifier current （Ikdr）, tWO of which were isolated pharmacologically with 10 mmol/L tetraethylammonium or 5 mmol/L 4-aminopyridine. Contamination of calcium-dependent potassium currents was minimized with no caldum and 0.2 mmol/L CdCl2 in an external solution. RESULTS： GST （10-100 μmol/L） reversibly and dosedependently reduced the peak amplitude of Ikto with an IC50 value of 22.0±6.9 μmol/L. To a lesser extent, Ikdr, was also inhibited in both peak current and sustained current. GST could not totally block the outward potassium current as a fraction of the outWard potassium current, which was insensitive to GST. GST had no effect on the steady-state activation （n =6） and inactivation kinetics （n =6） of Ikto. Sodium orthovanadate （1 retool/L）, a potent inhibitor of tyrosine phosphatase, significantly inhibited GST-induced inhibition （P〈 0.05）. CONCLUSION： GST can dose-dependently and reversibly block voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.

关键词： protein tyrosine kinase Patch-clamptechnique Genistein Voltage-gated potassium channel Colon Smooth muscle cells

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Signal transduction of bombesin-induced circular smooth muscle cell contraction in cat esophagus

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World Journal of Gastroenterology 2006年第14期12卷 2259-2263页

作者： Sung-Uk Park Chang-Yell Shin Jung-Su Ryu Hyen-O La Sun-Young Park Hyun-Ju Song Young-Sil Min Dong-Seok Kim Uy-Dong Sohn Department of Pharmacology College of Pharmacy Chung Ang UniversitySeoul 156-756Republic of Korea Department of Pharmacology College of Pharmacy Chung Ang UniversitySeoul 156-756Republic of Korea Research LaboratoryDong-A Pharm. Co. LtdYongin 449-905Kyunggi-DoRepublic of Korea Department of Pharmacology College of Medicine The Catholic University of KoreaSeoul 137-701Republic of Korea Research Division for Human Life Sciences Seoul National University 28 Yongon-DongChongno-GuSeoul 110-744Republic of Korea

AIM： To investigate the mechanism of bombesin-induced circular smooth muscle cell contraction in cat esophagus. METHODS： Specific G protein or phospholipase C involved in cat esophagus contraction was identified, muscle cells were permeabilized with saponin. After per- meabilization of muscle cells, the Gi3 antibody inhibited bombesin-induced smooth muscle cell contraction. RESULTS： Incubation of permeabilized circular muscle cells with PLC-β3 antibody could inhibit bombesin-induced contraction. H-7, chelerythrine （PKC inhibitor） and genistein （protein tyrosine kinase inhibitor） inhibited bombesin-induced contraction, but DAG kinase inhibitor, R59949, could not inhibit it. To examine which mitogen-activated protein kinase （MAPK） was involved in bombesin-induced contTaction, the specific MAPK inhibitors （MEK inhibitor, PD98059 and p38 MAPK inhibitor, SB202190） were used. Preincubation of PD98059 blocked the contraction induced by bombesin in a concentration-dependent manner. However, SB202190 had no effects on contraction. CONCLUSION： Bombesin-induced circular muscle cell contraction in cat esophagus is mediated via a PKC or a PTK-dependent pathway or p44/p42 HAPK pathway.

关键词： Bombesin G protein Phospholipase C protein kinase C protein tyrosine kinase MAP kinase Cell contraction

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Expression,Purification,and Initial Characterization of GST-fusion v-SRC from a Bacterial Expression System

Expression,Purification,and Initial Characterization of GST-...

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首届长三角科技论坛——长三角生物医药发展论坛

作者：龚兴国钟文涛谢捷周远 nstitute of Biomacromolecule & Enzyme Engineering College of Life SciencesZhejiang UniversityHangzhou310027China Institute of Biomacromolecule & Enzyme Engineering College of Life SciencesZhejiang UniversityHangzhou310027China

v-Src is a non-receptor protein tyrosine kinase,which is involved in many signal transduct-ion pathways and closely related to the activation and/or development of *** we presentedthe expression,purification and initial characterization of GST-fusion v-Src in plsmid pLysS from abaterial expression *** culture conditions were examined in the indution of IPTG,andGSH affinity chromatography was used to purified v-Src *** the versatile sub-strate poly-(Glu:Tyr)4:1,ELISA was employed to ditermine the phosphorylation kinase activity of *** strategy seems to be more promise than the insect cell system or other eukaryotic systemsemployed in earlier Src expression.

关键词： v-src GST-fusion inclusion body orthogonalization protein tyrosine kinase

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Modulatory Effect of Rg_1 on the Activity of proteintyrosine kinase of Lymphocytes in the Elderly

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Journal of Chinese Pharmaceutical Sciences 1996年第1期5卷 7-12页

作者：刘俊达王舒刘红涛卫生部北京老年医学研究所北京医科大学免疫学教研室

The effect of Rg1,a saponin extracted froin Panax ginseng, on the phenotype,receptor and the activity of protein tyrosine kinase (PTK) of lymphocytes isolated from 7 healthy oldpersons were studied. The CD25, CD45RA and CD45RO phenotypes of lymphocytes were 4eter-mined by indirect immunofluorescence technique. The percentage of CD25, CD45RA and CD45ROpositive lymphocytes was 38.3%±17.3%, 46.0% 15.1%, and 52.6%±14.1% respectively after incu-bation with PHA (5 μ±/ml) for 72 hours. However, there were 58.0%±12.5%, CD25, 64.1% ± 12.4%,CD45RA, and 74.0%±8.0%, CD45RO positive cells in the presence of Rg, ( 1μg/ml) along with PHA(5 μg/ml) over the sanie period of incubation. A significant increase was induced by Rgi (P<0.05).The activities of PTK in the cytoplasm and membrane of lymphocytes were measured by ELISAmcthod after incubation with PHA or PHA+Rg1. The absorbance value of PTK activity in cytoplasmafter 72 hr incubation was 0. 120±0.020 in PHA group, but 0. 1 38±0.015 in PHA+Rg1 group. In thelymphocyte membrane, it was 0.374± 0.060 in PHA group and 0.403 ± 0.008 in PHA+Rg1 group(P<0.001). These results showed that Rgi significantly arid simultaneously increased both the PT Kactivity and the expression of phenotype of lymphocytes.

关键词： Lymphocyte Rg1 Receptor protein tyrosine kinase

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