Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl

A novel C3G isoform, designated p87C3G, lacking the most amino terminal region of the cognate protein has been found to be overexpressed in two CML cell lines, K562 and Boff 210, both expressing Bcr-Abl p210. p87C3G expression is also highly augmented in patients diagnosed with chronic myeloid leukemia (CML) Ph+, in comparison with healthy individuals, and returns to basal levels after treatment with STI571. p87C3G coimmunoprecipitates with both CrkL and Bcr-Abl in CML cell lines and coimmunoprecipitation between p87C3G and Bcr-Abl was also detected in primary cells from CML patients. These interactions have been confirmed by in vitro pull down experiments. The interaction between p87C3G and Bcr-Abl involves the SH3-binding domain of p87C3G and the SH3 domain of Abl and depends mostly on the first polyproline region of p87C3G. Furthermore, we also demonstrated that p87C3G is phosphorylated in vitro by a Bcr-Abl-dependent mechanism. These results indicate that p87C3G overexpression is linked to CML phenotype and that p87C3G may exert productive functional interactions with Bcr-Abl signaling components suggesting the implication of this C3G isoform in the pathogenesis of chronic myeloid leukemia. © 2005 Elsevier Inc. All rights reserved.This work was supported by grants SA17/02 and SA013/02 from Junta de Castilla y León, SAF2003-04177 and GEN2003- 20239-C06-02 from the Spanish Ministry of Education, FISFEDER PI021570 and FIS-FEDER PI030651 from the Spanish Ministry of Health. J.G., E.C. and S.M.-E. are predoctoral fellows supported by the Spanish Ministry of Education. J.M.H. was supported by a Biomedicine Research Grant from Sacyl, Junta de Castilla y Leon, Spain. C.G. was supported by the Ramón y Cajal Program from the Spanish Ministry of Education.Peer Reviewe

C3G silencing enhances STI-571-induced apoptosis in CML cells through p38 MAPK activation, but it antagonizes STI-571 inhibitory effect on survival

In this work we report evidences of a functional relationship between C3G and p38 MAPK in the apoptotic effect of STI-571 on the chronic myeloid leukemia (CML) cell line K562. This has been demonstrated by knocking down C3G and p38α using the interfering RNA approach, as well as through targeting p38 by its inhibitor SB203580. The results indicate that p38 is a mediator of the STI-571-induced apoptosis, while C3G plays a negative role on STI-571-mediated p38 activation through a Rap1-dependent mechanism. According to this, gene expression analysis in C3G silenced cells revealed an upregulation of a large number of genes involved in apoptosis. Some of these genes are also down-regulated (at the protein level) upon p38α knock-down, which further suggests a functional association between these two proteins. On the other hand, C3G knock-down reverts the STI-571-inhibitory effect on ERKs and Akt pathways in a Rap1-independent fashion. Moreover, C3G overexpression also increased both, basal and STI-571-induced apoptosis, in agreement with previous reports. Therefore, our results strongly suggest a dual regulatory role for C3G in CML cells, modulating both apoptosis and survival via Rap-dependent and independent mechanisms. © 2009 Elsevier Inc. All rights reserved.This work was supported by grants FIS-PI041324 and FIS-PI070078 (AP: FIS-PI041131 and FIS-PI070071) from ISCIII, Ministry of Health, Spain; GR93 from Junta de Castilla y León, Spain, as well as institutional support from Red Temática de Investigación Cooperativa en Cáncer (RD07/0020/0000) from ISCIII, Ministry of Health, Spain. V.M is a predoctoral student supported by grant FIS-PI070078. A.G.-U. is a predoctoral student supported by Comunidad Autónoma de Madrid, Spain.Peer Reviewe

但馬国 持河内村 金札2歩預り

日本銀行金融研究所所蔵藩札等資料番号：ⅢAエドb2-43-46-1科学研究費助成事業（研究成果公開促進費）で電子化を実施データベースの名称：藩札等に関する統合データベース課題番号：20HP8030利用に関するお問い合わせ：画像の転載（出版物・HP等）に際しては、日本銀行貨幣博物館への申請手続きが必要です。詳しくは貨幣博物館ホームページ（http://www.imes.boj.or.jp/cm/service/）をご覧ください

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GδCat (tgC3GδCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GδCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GδCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.The authors would like to thank Dr. Katya Ravid (Boston University School of Medicine) for kindly providing PF4-Globin plasmid. We also thank Raquel Hernández (Department of Hematology, Hospital Universitario de Salamanca) for her assistance in the aggregometry studies, and to Isabel Ramos for assistance with the mice colony.Peer Reviewe

C3G forma complejos con Bcr-Abl y p38αMAPK en las adhesiones focales en células de leucemia mieloide crónica. Implicaciones en la regulación de la adhesión de las células leucémicas

Resumen del trabajo presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Madrid (España) del 3 al 6 de septiembre de 2013.C3G y Bcr-Abl forman complejos con las proteínas de adhesión focal CrkL, p 130Cas, Cbl y Abi1 a través de interacciones SH3/SH3-b. La asociaci6n entre C3G y Bcr-Abl disminuye en células de leucemia mieloide crónica (LMC) K562 silenciadas para Abi1 Y p130Cas, lo que sugiere que Abil y pl30Cas son muy relevantes en esta interacción. Por otra parte, el silenciamiento de C3G, Abi1 o Cbl disminuye la adhesión a la fibronectina, mientras que eI silenciamiento de p 130Cas la aumenta. EI análisis mediante arrays de dominios SH3 indican que los dominios SH-b de C3G, Cbl y p130Cas interaccionan directamente con proteínas comunes relacionadas con la regulación de la adhesión y migración celular. Estudios de inmunoprecipitación e inmunofluorescencia revelan que C3G forma complejos con proteínas de las adhesiones focales como la paxilina y FAK y sus formas fosforiladas. Además, C3G, Abil, Cbl y p130Cas regulan la expresión y fosforilación de la paxilina y FAK. Asimismo, p38alfa MAPK también participa en la regulación de la adhesión las células de LMC, interaccionando con C3G, CrkL, FAK y paxilina y regulando la expresión de paxilina, CrkL e integrina alfa5, así como la fosforilación de paxilina. Por otra parte, el doble silenciamiento C3G/p38alfa disminuye la adhesión a fibronectina, de manera similar al silenciamiento individual de cada uno de estos genes. Estos datos sugieren que C3G y p38alfa MAPK actúan a través de una ruta común que regula la adhesión celular en células K562, tal Y como se describió anteriormente en la regulaci6n de la apoptosis. Nuestros resultados indican que la ruta C3G-p38alfaMAPK regula la adhesi6n celular en K562 a través de la interacción con proteínas de las adhesiones focales y Bcr-Abl, modulando la formación de diferentes complejos proteicos de las adhesiones focales.Peer Reviewe