Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a 'non-protein kinase C' phorbol ester receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.This work was supported by grant RO1-CA74197 (National Institutes of Health) and RPG 97-092-06-CNE (American Cancer Society).Peer Reviewe

β2-quimerina regula la adhesión celular mediada por E-cadherina

Resumen del póster presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Valencia del 7 al 10 de septiembre.β2-quimerina es una proteína GAP (GTPase Activating Protein) que regula específicamente la actividad de la GTPasa Rac. Rac tiene un papel fundamental en el control una gran variedad de procesos celulares incluyendo la proliferación, supervivencia, adhesión y la motilidad celular. La mayor parte de las funciones reguladas por Rac se ejecutan a través del control que esta GTPasa ejerce sobre el citoesqueleto celular. La actividad de Rac esta regulada por proteínas activadoras, denominadas GEF (Guanine Exchange Factors) y proteínas inhibidoras, que incluyen a los denominados GDI (Guanine Disociation Inhibitors) y a las proteínas GAP, entre las que se encuentra β2-quimerina. Se ha demostrado que β2-quimerina puede controlar la proliferación celular regulando el ciclo celular a través de la inhibición de Rac. Sin embargo, el papel de β2-quimerina en las funciones asociadas al control del citoesqueleto de actina ejercido por Rac son prácticamente desconocidas. Para determinar este papel hemos generado una línea celular que expresa de manera estable β2-quimerina (células MCF7-β2-quimerina), en la que hemos analizado el citoesqueleto de actina así como distintos componentes implicados en la adhesión celular. Nuestros resultados demuestran que la expresión de β2-quimerina altera la morfología celular y la expresión de E-cadherina, sin afectar la formación de focos de adhesión ni otros componentes de las uniones adherentes y uniones estrechas. La alteración de los niveles de E-cadherina debidos a la expresión de β2-quimerina no impide la formación de las uniones intercelulares, pero reduce de manera notable la firmeza de dichas uniones. Futuros estudios determinarán si esta función es relevante en procesos como la metástasis, en los que una disminución de las uniones célula-célula favorece la adquisición de fenotipos más invasivos.Peer Reviewe

ß2-chimaerin subcellular localization modulates Rac-mediated responses

Resumen del póster presentado al 22nd IUBMB & 37th FEBS Congress: "From Single Molecules to Systems Biology" celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.Chimaerins are a family of GTPase-activating proteins that inactivate the Rac GTPase in a diacylglycerol (DAG) dependent manner. This family is composed of four members, α1-and α2-chimaerin that originate from the CHN1 gene and ß1- and ß2-chimaerin which are products of the CHN2 gene. All chimaerin isoforms have a C1 domain, highly homologous to that of PKC isozymes and a catalytic GAP domain. α2- and ß2-chimaerins have also an N-terminal SH2 domain, most likely involved in heteromolecular interactions. According to the established model for chimaerin activation, it has been shown that EGF redistributes ß2-chimaerin to promote its association with Rac at the plasma membrane. In addition, ß2-chimaerin shows a perinuclear and Golgi localization in response to PMA treatment or after stimulation with EGF. These data suggest that the localization of chimaerins in different subcellular compartments may serve as a mechanism that regulates Rac responses in localized regions of the cell. To study the functional effect of the different cellular localizations of chimaerins we have analyzed the activity of ß2- chimaerin that we have artificially targeted to cell membrane, Golgi and endoplasmic reticulum. Our preliminary results indicate that ß2-chimaerin localized at the plasma membrane participate in the control of actin cytoskeleton and alters cell adherence. This effect is specific of ß2-chimaerin localized at the lipid raft, while targeting ß2-chimaerin to the disordered membrane does not impact on actin cytoskeleton organization. These preliminary data suggest that chimaerins subcellular localization determines the biological outcome of Rac site-specific signals.Peer Reviewe

The Rac-GAP β2-chimaerin has a dual function in breast carcinogenesis

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.β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer. Despite the relevance of this function, the precise contribution of β2-chimaerin to this pathology has been largely unknown. We show evidence of a differential role of β2-chimaerin in the migration and invasion of breast cancer cells depending on whether they are epithelial or mesenchymal. Expression of β2-chimaerin in LM2 cells, a mesenchymal-like breast cancer cell line, significantly reduced cell migration (analyzed by wound healing assays) and invasion (analyzed by trasnswell assays). However, expression of β2-chimaerin in the epithelial breast cancer cell line MCF7 had no effect in the migratory and invasive properties of these cells. This cell context dependency of the effects of β2-chimaerin raised an important question about the exact role of β2-chimaerin in breast cancer, a pathology that arises from epithelial cells that switch to a mesenchymal phenotype during cancer progression. To answer this question we analyzed the effect of the genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mouse model of breast cancer. We demonstrate that loss of β2-chimaerin accelerates tumor onset and, importantly, delays tumor progression, most likely by delaying the epithelial to mesenchymal transition of breast cancer cells. Finally, we show evidence that our findings can be extrapolated to the human pathology, since analysis of breast cancer databases shows that patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times.Financial support: JCYL (BIO/VA34/15).Peer Reviewe

Role of chimaerins, a group of Rac-specific GTPase activating proteins, in T-cell receptor signaling

Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that α2- and β2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling. In agreement with this, we have observed that α2- and β2-chimaerins translocate to the T-cell/B-cell immune synapse and, using both gain- and loss-of-function approaches, demonstrated that their catalytic activity is important for the inhibition of the T-cell receptor- and Vav1-dependent stimulation of the transcriptional factor NF-AT. Mutagenesis-based approaches have revealed the molecular determinants that contribute to the biological program of chimaerins during T-cell responses. Unexpectedly, we have found that the translocation of chimaerins to the T-cell/B-cell immune synapse does not rely on the canonical binding of diacylglycerol to the C1 region of these GTPase-activating proteins. Taken together, these results identify chimaerins as candidates for the downmodulation of Rac1 in T-lymphocytes and, in addition, uncover a novel regulatory mechanism that mediates their activation in T-cells. © 2007 Elsevier Inc. All rights reserved.This work was supported by grants to MJC from the Spanish Ministry of Health (PI052096), the Castilla-León Autonomous Government (SA051/04), the Mutua Madrileña Medical Foundation, and the Solórzano Foundation, to XRB from the US National Cancer Institute/NIH (5R01-CA73735-11), the Spanish Ministry of Education and Science (MES) (SAF2006-01789), the Castilla-León Autonomous Government (SA053A05), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0001, Spanish Ministry of Health), and to BA from the Spanish Ministry of Education and Science (SAF2005- 00937). MJC is an investigator of the Ramón y Cajal Program of the Spanish Ministry of Education and Science who is associated to the University of Salamanca.Peer Reviewe

The Rac GTPase in cancer: From old concepts to new paradigms

Rho family GTPases are critical regulators of cellular functions that play important roles in cancer progression. Aberrant activity of Rho small G-proteins, particularly Rac1 and their regulators, is a hallmark of cancer and contributes to the tumorigenic and metastatic phenotypes of cancer cells. This review examines the multiple mechanisms leading to Rac1 hyperactivation, particularly focusing on emerging paradigms that involve gain-of-function mutations in Rac and guanine nucleotide exchange factors, defects in Rac1 degradation, andmislocalization of Rac signaling components. The unexpected pro-oncogenic functions of Rac GTPase-activating proteins also challenged the dogma that these negative Rac regulators solely act as tumor suppressors. The potential contribution of Rac hyperactivation to resistance to anticancer agents, including targeted therapies, as well as to the suppression of antitumor immune response, highlights the critical need to develop therapeutic strategies to target the Rac pathway in a clinical setting.M.G. Kazanietz's laboratory is supported by grants R01-CA189765, R01-CA196232, and R01-ES026023 from the NIH. M.J. Caloca's laboratory has been partially supported by grants BIO/VA22/14, CSI090U14, and BIO/VA34/15 from the Castilla-León Autonomous Government (Spain).Peer Reviewe

Association of a novel polymorphism of the β2-chimaerin gene (CHN2) with smoking

[Objective]: The CHN2 gene encodes the β2-chimaerin, a Rac-specific guanosine-5′-triphosphatase activating protein with an important role in the establishment of functional brain circuitry by controlling axon pruning. Genetic studies suggest that the CHN2 gene harbors variants that contribute to addiction vulnerability and smoking behavior. To further evaluate the role of β2-chimaerin in nicotine addiction, we investigated the association of 3 individual polymorphisms of the CHN2 gene with smoking dependence. [Methods]:Three hundred sixty-one healthy volunteers, 173 smokers (mean ± SD age, 60.4 ± 1.4 years) and 188 control subjects (mean ± SD age, 45.9 ± 1.4 years) were genotyped for 3 single-nucleotide polymorphisms in the CHN2 gene (rs3750103, rs12112301, and rs186911567). The association of these polymorphisms with smoking habits was analyzed. [Results]:There was no significant association of polymorphisms rs12112301 and rs3750103 with smoking. However, there was a significant difference in the frequency of the rs186911567 polymorphism between the smokers and the controls (P = 0.003).[Conclusions]: We report for the first time a significant association of the novel rs186911567 polymorphism of the CHN2 gene with smoking.This work was supported by the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias, PI052096 to MJC, PI10/00219 to RGS), the Spanish Ministry of Science and Innovation cosponsored by the plan E (BFU2009-08051 to MJC), and the Castilla y Leon Autonomous Government (BIO103/VA44/11 to MJC).Peer Reviewe

A new role of the Rac-GAP ß2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.This work was initially supported by a grant from the Spanish Ministry of Economy and Competitiveness to MJC (BFU2009-08051), but could be finished only thanks to the support from the Castilla-León Autonomous Government to MJC (grants BIO103/VA44/11, BIO/VA22/14, CSI090U14 and BIO/VA34/15).Peer Reviewe

Inverted signaling hierarchy between RAS and RAC in T-lymphocytes

In order to generate coherent biological responses to extracellular stimuli, cells have established synergistic and antagonistic crosstalk between pathways with similar or opposing functions, respectively. Two routes cooperating in the generation of mitogenic and cytoskeletal functions are those induced by Ras and Rho/Rac GTPases. In these signaling interactions, Rho/Rac proteins have been always placed in a downstream position respect to Ras in all cell systems analysed so far. In this report, we describe that such signaling hierarchy does not apply to T-lymphocytes. Thus, we show that both Rac1 GDP/GTP exchange factors such as Vav and constitutively active versions of Rac1 can promote the effective stimulation of the Ras pathway in T-lymphocytes. The molecular link for this new type of pathway interconnectivity is RasGRP1, a diacylglycerol-dependent GDP/GTP exchange factor for Ras that translocates to the plasma membrane in a Vav- and Rac1-dependent manner. The effect of the Vav/Rac1 pathway on the Ras pathway is highly dependent on the activity of phospliolipase C-γ, the key cellular supplier of intracellular diacylglycerol. Signaling experiments suggest that this crosstalk represents a signaling strategy used by the T-cell receptor to promote robust biological responses of both the Rac/Rho and Ras pathways upon antigen engagement.We thank M Blázquez for technical assistance. This work was supported by the US National Cancer Institute (CA7373501), the Programa General del Conocimiento of the Spanish Ministry of Science and Technology (PM99-0093), and a grant from the Ministry of Education and Culture of the Autonomous Government of Castilla- León (SA051/02). MJC and JLZ are investigators of the Ramón y Cajal Program (Spanish Ministry of Science and Technology) associated to the University of Salamanca. The Centro de Investigación del Cáncer is supported by endowments from the CSIC, University of Salamanca, Castilla-León Autonomous Government, the Spanish National Network of Cancer Centers (Carlos III Institute, Spanish Ministry of Health), the Foundation for Cancer Research of Salamanca (FICUS), and the Solórzano and Moraza Foundations.Peer Reviewe

Rho GTPases and their regulators in addiction: A focus on the association of a ß2-chimaerin polymorphism with smoking

Tobacco addiction is a complex disorder that involves multiple molecular mechanisms and is influenced by genetic factors. Repeated exposure to nicotine induces the remodeling of synaptic connections, a process that contributes to the long-lasting nature of tobacco addiction. Rho GTPases are key regulators of synaptic structure and function and, therefore, these proteins have a potential role in nicotine addiction. In this chapter we will briefly describe some studies that identify genes encoding Rho GTPases and their regulators as candidate genes for smoking-related behaviors, with special focus on the CHN2 gene. The CHN2 gene encodes the β2-chimaerin, a member of the chimaerin family of GTPase activating proteins that selectively inactivates the GTPase Rac. Genetic studies suggest that nucleotide variants of the CHN2 gene may influence success in smoking cessation. In addition, work has identified a significant association of the rs186911567 polymorphism in the CHN2 gene with smoking in a Spanish population. We discuss how this polymorphism could alter β2-chimaerin function and the signaling pathways mediated by this protein that could influence smoking behavior.Peer Reviewe