Small GTPase Rac1: Structure, Localization, and Expression of the Human Gene
Rac1 is a member of the Rho family of small GTPases involved in signal transduction pathways that control proliferation, adhesion, and migration of cells during embryonic development and invasiveness of tumor cells. Here we present the complete structure of the human RAC1 gene and characterize its expression. The gene comprises 7 exons over a length of 29 kb and is localized to chromosome 7p22. The GC-rich gene promoter shows characteristics of a housekeeping gene and Northern blot studies revealed ubiquitous expression of two rac1 transcripts, 1.2 and 2.5 kb in size. The two transcripts are expressed in tissue-specific ratios, reflecting competition between two alternative polyadenylation sites. The RAC1 but not RAC2 gene contains an additional exon 3b that is included by alternative splicing into the variant Rac1b, a constitutively active mutant which induces the formation of lamellipodia in fibroblasts. These data indicate that the RAC1 gene encodes two signaling GTPases. The gene structure reported here will enable studies on the regulation of RAC1 expression during tumorigenesis and development.
Available from: Nishigandha Naik
- "The 25 kd band could be of rac1b. Rac1b acts like a fast cycling GTPase  and induces formation of lamellipodia in NIH3T3 . Similarly in normal PMNL too, rac1b could be responsible for actin polymerization in lamellipodia. "
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ABSTRACT: Chronic Myeloid Leukemia (CML) is a malignant pluripotent stem cells disorder of myeloid cells. In CML patients, polymorphonuclear leukocytes (PMNL) the terminally differentiated cells of myeloid series exhibit defects in several actin dependent functions such as adhesion, motility, chemotaxis, agglutination, phagocytosis and microbicidal activities. A definite and global abnormality was observed in stimulation of actin polymerization in CML PMNL. Signalling molecules ras and rhoGTPases regulate spatial and temporal polymerization of actin and thus, a broad range of physiological processes. Therefore, status of these GTPases as well as actin was studied in resting and fMLP stimulated normal and CML PMNL.
To study expression of GTPases and actin, Western blotting and flow cytometry analysis were done, while spatial expression and colocalization of these proteins were studied by using laser confocal microscopy. To study effect of inhibitors on cell proliferation CCK-8 assay was done. Significance of differences in expression of proteins within the samples and between normal and CML was tested by using Wilcoxon signed rank test and Mann-Whitney test, respectively. Bivariate and partial correlation analyses were done to study relationship between all the parameters.
In CML PMNL, actin expression and its architecture were altered and stimulation of actin polymerization was absent. Differences were also observed in expression, organization or stimulation of all the three GTPases in normal and CML PMNL. In normal PMNL, ras was the critical GTPase regulating expression of rhoGTPases and actin and actin polymerization. But in CML PMNL, rhoA took a central place. In accordance with these, treatment with rho/ROCK pathway inhibitors resulted in specific growth inhibition of CML cell lines.
RhoA has emerged as the key molecule responsible for functional defects in CML PMNL and therefore can be used as a therapeutic target in CML.
Molecular Cancer 03/2012; 11(1):16. DOI:10.1186/1476-4598-11-16 · 4.26 Impact Factor
Available from: Xilin Chen
- "and H 2 O 2 , in the regulation of the expression of inflammatory genes remains unclear. Rac1 is a member of the Rho family of small GTPases involved in signal transduction pathways that control proliferation , adhesion, and migration of cells during embryonic development and invasiveness of tumor cells (Matos et al., 2000). In phagocytic cells, Rac proteins are involved in the assembly of the neutrophil NADPH oxidase system and responsible for transferring electrons from NADPH to molecular oxygen with the subsequent production of O 2 . "
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ABSTRACT: Oxidative signals play an important role in the regulation of endothelial cell adhesion molecule expression. Small GTP-binding protein Rac1 is activated by various proinflammatory substances and regulates superoxide generation in endothelial cells. In the present study, we demonstrate that adenoviral-mediated expression of dominant negative N17Rac1 (Ad.N17Rac1) suppresses tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin gene expression in a dose-dependent manner. Ad.N17Rac1 did not inhibit TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) binding activity or inhibitor of NF-kappaB-alpha degradation. In contrast, Ad.N17Rac1 inhibited TNF-alpha-induced NF-kappaB-driven HIV(kappaB)(4)-CAT and p288VCAM-Luc promoter activity, suggesting that N17Rac1 inhibits TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 through suppressing NF-kappaB-mediated transactivation. In addition, expression of superoxide dismutase by adenovirus suppressed TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 mRNA accumulation. However, adenoviral-mediated expression of catalase only partially inhibited TNF-alpha-induced E-selectin gene expression and had no effect on VCAM-1 and ICAM-1 gene expression. These data suggest that Rac1 and superoxide play crucial roles in the regulation of expression of cell adhesion molecules in endothelial cells.
Journal of Pharmacology and Experimental Therapeutics 06/2003; 305(2):573-80. DOI:10.1124/jpet.102.047894 · 3.97 Impact Factor
Available from: archives-ouvertes.fr
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ABSTRACT: The thiopurine drugs, azathioprine, 6-mercaptopurine and 6-thioguanine, have been used for decades for their cytotoxic and immunosuppressive properties in the treatment of leukemias, chronic inflammatory or autoimmune diseases and in the prevention of allograft rejection. However, some patients treated with conventional doses of these molecules develop very severe side effects. The deficient activity of the thiopurine S-methyltransferase (TPMT), an enzyme involved in thiopurine inactivation, is one of the key factors in the myelotoxicity of these drugs. The determination of the TPMT phenotype by genotyping test is a preventive measure before the initiation of thiopurine therapy and is based on the identification of the most common inactivating mutations of the TPMT gene. First, our study consisted in the functional analysis of four new allelic variants of TPMT, using an heterologous expression system, the yeast S. cerevisiae. The non-functional character of two of those variants was demonstrated. However, TPMT deficiency explain only about 30 % of cases of thiopurine myelotoxicity, suggesting the existence of other genetic abnormalities affecting other genes involved in the response toward thiopurines. Accordingly, we studied the genetic polymorphism of two other proteins, the inosine monophosphate dehydrogenase type 2 (IMPDH2), a key enzyme in the production of the active metabolites of thiopurine, and the RhoGTPase RAC1, which is one of the pharmacological targets of these molecules. Some of the polymorphisms that we identified in those two genes seem to affect in vitro the expression and / or activity of these proteins and, therefore, could contribute to inter-individual variations of the response to thiopurine
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