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Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., C3G is an activator of members of the Rap subfamily of Ras proteins. Several studies suggest that C3G plays a role in different processes of differentiation acting through its main target Rap1. C3G is induced during neural differentiation and monocytic differentiation to macrophages. Similarly, an increase in C3G protein levels and its association with Crk is required for adipocyte differentiation. There are also evidences supporting the involvement of C3G in the differentiation of megakaryocytes to platelets through Rap1 activation, although its function in early stages of megakaryocytic differentiation has not yet been addressed. Using transgenic mouse models for C3G and C3GΔCat (C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 gene promoter, we have found that C3G plays a role in the differentiation of immature hematopoietic cells to megakaryocytes. Bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased CD41 and CD61 expression upon thrombopoietin (TPO) treatment. Polyploidization analysis supports the hypothesis of a positive role of C3G in megakaryocytic differentiation. Thus, C3G overexpression increased the levels of CD41+ megakaryocytes with 16N and 32N ploidy. In addition, preliminary studies using fresh bone marrow explants, incubated with Tyrode´s Buffer containing 5% mouse serum, showed an increased migration from the osteoblastic niche to the vascular niche and an enhanced ability to form pro-platelets in C3G transgenic megakaryocytes, as compared to wild-type ones. These results suggest the participation of C3G in different key aspects of megakaryopoiesis by a mechanism mediated by its GEF activity.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., C3G is a guanine nucleotide exchange factor (GEF) of several proteins within the Ras superfamily, including Rap1a/b and R-Ras. Rap1 is known to be involved in critical aspects of platelet function, including aggregation, adhesion and spreading, through the activation of the platelet integrin αIIbβ3. Using transgenic mouse models expressing C3G or C3GΔCat (a mutant lacking the catalytic domain) in megakaryocytes and platelets, our group has identified C3G as a mediator of Rap1 actions leading to platelet activation and aggregation. Furthermore, C3G promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface, following its activation. In addition, preliminary immunofluorescence analysis suggests that C3G modulates the release of VEGF and endostatin from thrombin-activated platelets. The goal of the present study was to further characterize the underlying mechanisms that regulate the function of C3G in the differential release of pro- and anti-angiogenic factors upon platelet activation, and to evaluate the angiogenic potential of the platelet releasate. We have found that VEGF release was up-regulated in thrombin-activated mouse platelets from both, C3G and C3GΔCat, transgenic mice. Additionally, thrombospondin release was also up-regulated in the latter. The releasate of TgC3G platelets had an overall pro-angiogenic effect as evidenced in a capillary-tube formation assay with HUVECs and in heterotopic tumor mouse models using B16 and 3LL cells. On the other hand, platelet spreading analysis revealed an up-regulation of this function in both transgenic mouse platelets upon thrombin activation, with TgC3G platelets showing the highest extension area. A preliminary co-IP analysis revealed an interaction between C3G and VAMP-7 upon activation, which could explain this result. Taken together, our data indicate the co-existence of RapGEF-dependent and independent mechanism through which C3G is regulating platelet secretion, and suggest an overall pro-angiogenic effect of platelet C3G.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., C3G is guanine nucleotide exchange factor for Rap1b, a protein of the Ras family involved in critical aspects of platelet function through the activation of integrin aIIbb3. Using transgenic mouse models, with specific expression in platelets, of full length C3G (tgC3G) or mutant lacking the GEF domain (tgC3GDCat), our group has unveiled an important role of C3G in primary hemostasis. Thus, C3G-Rap1 participates in thrombin-triggered platelet activation, aggregation and clotting through a PKC-mediated pathway. In addition, C3G-overexpressing platelets also show a stronger response to ADP, as compared to wt platelets. Based on these results, we have deepened into the role of C3G in platelet signaling by using several inhibitors of different platelets signaling pathways. This includes: clopidogrel and 2MeSAMP (for P2Y12 ADP receptor), MRS2179 (for P2Y1 ADP receptor), SB203580 (for p38a/b MAPK), U0126 (for ERK1/2), aspirin (for TXA2 synthesis) and wortmannin (for PI3K). We have performed activation, aggregation, and Rap1 activity assays in platelets stimulated with thrombin or ADP/fibrinogen in the presence or absence of the above referred inhibitors. Our results indicate that thrombin- or ADP-stimulated tgC3G platelets are more sensitive to the action of clopidogrel, SB203580, U0126 and aspirin, as compared to wild type platelets. In contrast, tgC3GDCat platelets are more resistant to these inhibitors than their corresponding control platelets. No differences were observed with the rest of inhibitors. These results suggest that C3G may participate in the activation of Rap1 mediated by ADP-P2Y12, but independent of PI3K. Furthermore, our results support a role for C3G in the thromboxane synthesis pathway, probably through modulating ERKs and p38a MAPK activities.

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015., C3G (Crk-SH3-binding Guanine nucleotide Exchange Factor) is an activator of Ras family member Rap1/2. Several studies suggest a role for C3G in cell differentiation; C3G is induced during neural differentiation, as well as during differentiation of monocytic cells to macrophages. Similarly, enhancement in C3G protein and its association with Crk is required for adipocyte differentiation. Some evidences suggest a participation of C3G in the differentiation of megakaryocytes to platelets through Rap1 activation, although its potential role in early stages of megakaryocytic differentiation has not been addressed yet. Using erythroleukemic cell lines K562 and HEL we have found that C3G plays a role in the differentiation of immature cells to megakaryocytes, as overexpression of C3G increases the expression of megakaryocytic markers CD41 and CD61, while decreases erythroid marker glycophorin A. In contrast, the expression of CD41/CD61 decreases in C3G silenced cells. Morphology and polyploidisation analysis support the hypothesis of a positive role of C3G in megakaryocytic differentiation. This effect of C3G in megakaryopoiesis is dependent on PKC pathway as bisyndolylmaleimide completely abrogated it. Additionally, studies in double C3G/p38aMAPK K562 knockdown clones suggest a relationship between these two proteins in the regulation of K562 megakaryocytic differentiation. In concordance with the studies in cell lines, TPOstimulated bone marrow cells from megakaryocyte- and platelet-specific C3G transgenic mice showed higher levels of CD41/CD61 as compared to wild type cells. In contrast, bone marrow cells from C3GDCat transgenic mice showed lower levels of these megakaryocytic markers, indicating that the effect of C3G in megakaryocytic differentiation is Rap1-dependent.

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.