{"id":{"repo_id":"ajou","oai_identifier":"oai:repository.ajou.ac.kr:201003/1488"},"canonical_url":"https://search.dev.ndltd.org/etd/ajou/oai:repository.ajou.ac.kr:201003/1488","repository":{"repo_id":"ajou","name":"Ajou University","base_url":"http://repository.ajou.ac.kr/oai/request"},"display":{"title":"Up-modulation of Hepatocyte Growth Factor/ c-Met Signaling by Protein Kinase C-g and its Mechanism","abstract":"Protein kinase C (PKC) family is composed of 11 isoenzymes, and has been known to down-modulate the tyrosine kinase activity of c-Met, the receptor for hepaotcyte growth factor (HGF). However, specified roles by PKC isoenzymes on the regulation of HGF/c-Met signaling have never been addressed yet. In this study, attempts have been made to investigate the effect of PKC-γ, an isoenzyme of PKC, on the HGF/c-Met signaling and its mechanism. In PKC-γ-transfected NIH3T3 cells, c-Met autophosphorylation level was increased compared to that of the normal NIH3T3 cells, which was partly abolished by the pre-treatment of Go¨6976; an inhibitor of PKC-α, β1 and γ. Also, the Erk phosphorylation level was significantly increased in these cells with the treatment of HGF. The expression levels of both c-Met mRNA and protein were not altered in PKC-γ transfected cells. Translocation of PKC-γ to the membrane after HGF treatment was not observed either. Interestingly, a significant amount of PKC-γ revealed to be constitutively localized at the membrane, unlike PKC-α. Furthermore, co-immunoprecipitation assay revealed that PKC-γ interacts with c-Met constitutively. Treatment with HGF does not affect the association of PKC-γ with c-Met or the tyrosine phosphorylation level of PKC-γ. Instead, the serine phosporylation level of c-Met was increased. To define phosphorylation site of c-Met by PKC-γ, we perform in vitro kinase assay using various GST fusion proteins with different domains of cytosolic Met. This result indicates that juxtamembrane domain of c-Met is directly phosphorylated by PKC-γ. It seems that the up-modulation of c-Met signaling in PKC-γ transfected NIH3T3 cells is not associated with HGF-induced rapid change but with the constitutive association of c-Met with PKC-γ and effect on c-Met activation via phosphorylation of specific serine1020 residue in juxtamembrane domain.","abstract_html":"Protein kinase C (PKC) family is composed of 11 isoenzymes, and has been known to down-modulate the tyrosine kinase activity of c-Met, the receptor for hepaotcyte growth factor (HGF). However, specified roles by PKC isoenzymes on the regulation of HGF/c-Met signaling have never been addressed yet. In this study, attempts have been made to investigate the effect of PKC-γ, an isoenzyme of PKC, on the HGF/c-Met signaling and its mechanism. In PKC-γ-transfected NIH3T3 cells, c-Met autophosphorylation level was increased compared to that of the normal NIH3T3 cells, which was partly abolished by the pre-treatment of Go¨6976; an inhibitor of PKC-α, β1 and γ. Also, the Erk phosphorylation level was significantly increased in these cells with the treatment of HGF. The expression levels of both c-Met mRNA and protein were not altered in PKC-γ transfected cells. Translocation of PKC-γ to the membrane after HGF treatment was not observed either. Interestingly, a significant amount of PKC-γ revealed to be constitutively localized at the membrane, unlike PKC-α. Furthermore, co-immunoprecipitation assay revealed that PKC-γ interacts with c-Met constitutively. Treatment with HGF does not affect the association of PKC-γ with c-Met or the tyrosine phosphorylation level of PKC-γ. Instead, the serine phosporylation level of c-Met was increased. To define phosphorylation site of c-Met by PKC-γ, we perform in vitro kinase assay using various GST fusion proteins with different domains of cytosolic Met. This result indicates that juxtamembrane domain of c-Met is directly phosphorylated by PKC-γ. It seems that the up-modulation of c-Met signaling in PKC-γ transfected NIH3T3 cells is not associated with HGF-induced rapid change but with the constitutive association of c-Met with PKC-γ and effect on c-Met activation via phosphorylation of specific serine1020 residue in juxtamembrane domain.","abstract_has_math":false,"creators":["김, 율"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["이, 재호","대학원 의학과","200324254"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-02-18T07:21:46Z","date_published":"2011-02-18T07:21:46Z","updated_at":"2026-07-24T00:51:22Z","subjects":["hepatocyte growth factor","c-Met","protein kinase C-γ","up-modulation"],"languages":["ko"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000207","000000000207"],"render_values":[{"text":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000207","href":"http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000207","code":true},{"text":"000000000207","href":null,"code":true}]}]},"links":{"outbound_url":"http://repository.ajou.ac.kr/handle/201003/1488","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["이, 재호","대학원 의학과","200324254","김, 율"]},{"key":"dc:creator","label":"Author","values":["김, 율"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-02-18T07:21:46Z","2005"]},{"key":"dc:type","label":"Dc Type","values":["Thesis","Theses"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["hepatocyte growth factor","c-Met","protein kinase C-γ","up-modulation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ko"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://repository.ajou.ac.kr/handle/201003/1488","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000207","000000000207"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Protein kinase C (PKC) family is composed of 11 isoenzymes, and has been known to down-modulate the tyrosine kinase activity of c-Met, the receptor for hepaotcyte growth factor (HGF). However, specified roles by PKC isoenzymes on the regulation of HGF/c-Met signaling have never been addressed yet. In this study, attempts have been made to investigate the effect of PKC-γ, an isoenzyme of PKC, on the HGF/c-Met signaling and its mechanism. In PKC-γ-transfected NIH3T3 cells, c-Met autophosphorylation level was increased compared to that of the normal NIH3T3 cells, which was partly abolished by the pre-treatment of Go¨6976; an inhibitor of PKC-α, β1 and γ. Also, the Erk phosphorylation level was significantly increased in these cells with the treatment of HGF. The expression levels of both c-Met mRNA and protein were not altered in PKC-γ transfected cells. Translocation of PKC-γ to the membrane after HGF treatment was not observed either. Interestingly, a significant amount of PKC-γ revealed to be constitutively localized at the membrane, unlike PKC-α. Furthermore, co-immunoprecipitation assay revealed that PKC-γ interacts with c-Met constitutively. Treatment with HGF does not affect the association of PKC-γ with c-Met or the tyrosine phosphorylation level of PKC-γ. Instead, the serine phosporylation level of c-Met was increased. To define phosphorylation site of c-Met by PKC-γ, we perform in vitro kinase assay using various GST fusion proteins with different domains of cytosolic Met. This result indicates that juxtamembrane domain of c-Met is directly phosphorylated by PKC-γ. It seems that the up-modulation of c-Met signaling in PKC-γ transfected NIH3T3 cells is not associated with HGF-induced rapid change but with the constitutive association of c-Met with PKC-γ and effect on c-Met activation via phosphorylation of specific serine1020 residue in juxtamembrane domain.","Protein kinase C (PKC) family는 11가지의 isoenzyme으로 이루어져 있고 hepatocyte growth factor receptor 인 c-Met의 tyrosine kinase activity를 down modulation 한다고 보고되어왔다. 하지만 HGF/c-Met의 신호 전달에 있어서 각각의 PKC isoenzyme이 어떠한 역할을 하는지는 아직 밝혀지지 않았다. 본 연구에서는 PKC isoenzyme 중 하나인 PKC-γ가 HGF/c-Met의 신호 전달에 미치는 영향과 그것의 기전을 연구하였다. 이전에 알려진 것과는 반대로 PKC-γ가 transfection된 NIH3T3 세포주에서 Met의 활성이 증가하는 up-modulation 현상을 최초로 관찰하였다. 이러한 Met의 autophosphorylation의 증가는 PKC-γ의 inhibitor인 Go6976에 의해서 저해되었다. 또한 PKC-γ가 transfection된 NIH3T3 세포주에서 Met의 downstream signaling molecule인 Erk의 인산화도 parental NIH3T3 cell에 비해 현저히 증가하였다. PKC-γ의 activity을 살펴보기 위하여 membrane으로의 translocation을 살펴본 결과, HGF 처리 후에 PKC-γ가 membrane으로 이동하는 현상은 관찰할 수 없었지만 많은 양의 PKC-γ가 PKC-α와는 다르게 membrane에 constitutive하게 위치하고 있었다. 또한 PKC-γ는 Met과 결합하고 있음을 co- assay로 확인하였다. PKC-γ가 어떠한 기전을 통해서 Met의 activity에 영향을 주는지 알아보기 위해서 PKC-γ/NIH3T3 세포주에서 Met을 immunoprecipitation하고 phospho-Serine 항체를 이용하여 살펴본 결과 Met의 serine이 인산화 되는 것을 볼 수 있었다. 따라서 PKC-γ가 Met의 serine을 인산화 시키는 자리를 찾기 위해서 Met의 cytosol 부분을 나누어 GST fusion protein을 제작하였다. In vitro kinase assay 결과 PKC-γ는 Met의 juxtamembrane 부분을 direct하게 인산화시킴을 알 수 있었고 이 부분의 peptide를 MALDI-TOF로 분석한 결과 serine1020 임이 확인되었다.","\"차례 국문요약 = ⅰ 차례 = ⅲ 그림차례 = ⅴ Ⅰ. 서론 = 1 Ⅱ. 재료 및 방법 = 4 A. 세포 배양 = 4 B. 항체 및 시약 = 4 C. 면역 침전(Immunoprecipitation)과 Western blotting = 5 D. 유전자 이입(transfection) = 5 E. 면역 형광 염색(immunocytochemstry) = 6 F. PKC-g Kinase assay = 6 G. GST fusion protein 제작 = 7 H. in vitro kinase assay = 8 I. GST pull down assay = 9 J. MALDI-TOF 분석 = 9 Ⅲ. 결과 = 10 A. PKC-g에 의한 c-Met의 autophosporylation level 증가 = 10 B. PKC-g에 의한 Erk phosporylation level의 증가 = 10 C. PKC- g와 c-Met의 결합 = 11 D. c-Met에 의한 PKC-g 활성화 = 12 E. PKC-g에 의한 c-Met의 활성화 = 13 F. PKC-g와 c-Met의 결합 부분 = 13 G. PKC-g에 의한 c-Met 의 juxtamembrane 부분의 인산화 = 14 H. PKC-g에 의한 c-Met의 serine1020의 인산화 = 14 Ⅳ. 고찰 = 28 Ⅴ. 결론 = 31 참고문헌 = 32 ABSTRACT = 37\"","Master"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Up-modulation of Hepatocyte Growth Factor/ c-Met Signaling by Protein Kinase C-g and its Mechanism","Protein Kinase C-γ 에 의한 c-Met 활성 증가의 기전"]}]}],"canonical_facts":{"dc:contributor":["이, 재호","대학원 의학과","200324254","김, 율"],"dc:creator":["김, 율"],"dc:date":["2011-02-18T07:21:46Z","2005"],"dc:description":["Protein kinase C (PKC) family is composed of 11 isoenzymes, and has been known to down-modulate the tyrosine kinase activity of c-Met, the receptor for hepaotcyte growth factor (HGF). However, specified roles by PKC isoenzymes on the regulation of HGF/c-Met signaling have never been addressed yet. In this study, attempts have been made to investigate the effect of PKC-γ, an isoenzyme of PKC, on the HGF/c-Met signaling and its mechanism. In PKC-γ-transfected NIH3T3 cells, c-Met autophosphorylation level was increased compared to that of the normal NIH3T3 cells, which was partly abolished by the pre-treatment of Go¨6976; an inhibitor of PKC-α, β1 and γ. Also, the Erk phosphorylation level was significantly increased in these cells with the treatment of HGF. The expression levels of both c-Met mRNA and protein were not altered in PKC-γ transfected cells. Translocation of PKC-γ to the membrane after HGF treatment was not observed either. Interestingly, a significant amount of PKC-γ revealed to be constitutively localized at the membrane, unlike PKC-α. Furthermore, co-immunoprecipitation assay revealed that PKC-γ interacts with c-Met constitutively. Treatment with HGF does not affect the association of PKC-γ with c-Met or the tyrosine phosphorylation level of PKC-γ. Instead, the serine phosporylation level of c-Met was increased. To define phosphorylation site of c-Met by PKC-γ, we perform in vitro kinase assay using various GST fusion proteins with different domains of cytosolic Met. This result indicates that juxtamembrane domain of c-Met is directly phosphorylated by PKC-γ. It seems that the up-modulation of c-Met signaling in PKC-γ transfected NIH3T3 cells is not associated with HGF-induced rapid change but with the constitutive association of c-Met with PKC-γ and effect on c-Met activation via phosphorylation of specific serine1020 residue in juxtamembrane domain.","Protein kinase C (PKC) family는 11가지의 isoenzyme으로 이루어져 있고 hepatocyte growth factor receptor 인 c-Met의 tyrosine kinase activity를 down modulation 한다고 보고되어왔다. 하지만 HGF/c-Met의 신호 전달에 있어서 각각의 PKC isoenzyme이 어떠한 역할을 하는지는 아직 밝혀지지 않았다. 본 연구에서는 PKC isoenzyme 중 하나인 PKC-γ가 HGF/c-Met의 신호 전달에 미치는 영향과 그것의 기전을 연구하였다. 이전에 알려진 것과는 반대로 PKC-γ가 transfection된 NIH3T3 세포주에서 Met의 활성이 증가하는 up-modulation 현상을 최초로 관찰하였다. 이러한 Met의 autophosphorylation의 증가는 PKC-γ의 inhibitor인 Go6976에 의해서 저해되었다. 또한 PKC-γ가 transfection된 NIH3T3 세포주에서 Met의 downstream signaling molecule인 Erk의 인산화도 parental NIH3T3 cell에 비해 현저히 증가하였다. PKC-γ의 activity을 살펴보기 위하여 membrane으로의 translocation을 살펴본 결과, HGF 처리 후에 PKC-γ가 membrane으로 이동하는 현상은 관찰할 수 없었지만 많은 양의 PKC-γ가 PKC-α와는 다르게 membrane에 constitutive하게 위치하고 있었다. 또한 PKC-γ는 Met과 결합하고 있음을 co- assay로 확인하였다. PKC-γ가 어떠한 기전을 통해서 Met의 activity에 영향을 주는지 알아보기 위해서 PKC-γ/NIH3T3 세포주에서 Met을 immunoprecipitation하고 phospho-Serine 항체를 이용하여 살펴본 결과 Met의 serine이 인산화 되는 것을 볼 수 있었다. 따라서 PKC-γ가 Met의 serine을 인산화 시키는 자리를 찾기 위해서 Met의 cytosol 부분을 나누어 GST fusion protein을 제작하였다. In vitro kinase assay 결과 PKC-γ는 Met의 juxtamembrane 부분을 direct하게 인산화시킴을 알 수 있었고 이 부분의 peptide를 MALDI-TOF로 분석한 결과 serine1020 임이 확인되었다.","\"차례 국문요약 = ⅰ 차례 = ⅲ 그림차례 = ⅴ Ⅰ. 서론 = 1 Ⅱ. 재료 및 방법 = 4 A. 세포 배양 = 4 B. 항체 및 시약 = 4 C. 면역 침전(Immunoprecipitation)과 Western blotting = 5 D. 유전자 이입(transfection) = 5 E. 면역 형광 염색(immunocytochemstry) = 6 F. PKC-g Kinase assay = 6 G. GST fusion protein 제작 = 7 H. in vitro kinase assay = 8 I. GST pull down assay = 9 J. MALDI-TOF 분석 = 9 Ⅲ. 결과 = 10 A. PKC-g에 의한 c-Met의 autophosporylation level 증가 = 10 B. PKC-g에 의한 Erk phosporylation level의 증가 = 10 C. PKC- g와 c-Met의 결합 = 11 D. c-Met에 의한 PKC-g 활성화 = 12 E. PKC-g에 의한 c-Met의 활성화 = 13 F. PKC-g와 c-Met의 결합 부분 = 13 G. PKC-g에 의한 c-Met 의 juxtamembrane 부분의 인산화 = 14 H. PKC-g에 의한 c-Met의 serine1020의 인산화 = 14 Ⅳ. 고찰 = 28 Ⅴ. 결론 = 31 참고문헌 = 32 ABSTRACT = 37\"","Master"],"dc:format":["application/pdf"],"dc:identifier":["http://repository.ajou.ac.kr/handle/201003/1488","http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000207","000000000207"],"dc:language":["ko"],"dc:subject":["hepatocyte growth factor","c-Met","protein kinase C-γ","up-modulation"],"dc:title":["Up-modulation of Hepatocyte Growth Factor/ c-Met Signaling by Protein Kinase C-g and its Mechanism","Protein Kinase C-γ 에 의한 c-Met 활성 증가의 기전"],"dc:type":["Thesis","Theses"]},"updated_at":"2026-07-24T00:51:22Z"}