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Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere

Michel Souchet1,*, Elodie Portales-Casamar2,*, David Mazurais1, Susanne Schmidt2, Isabelle Léger1, Jean-Luc Javré1, Philippe Robert1, Isabelle Berrebi-Bertrand1, Antoine Bril1, Bernard Gout1, Anne Debant2,{ddagger} and Thierry P. G. Calmels1,{ddagger}

1 SmithKline Beecham Laboratoires Pharmaceutiques, Unité de Biologie Cardiovasculaire, 4 rue du Chesnay Beauregard, BP 96205, 35760 Saint-Grégoire, France
2 Centre de Recherches en Biochimie Macromoléculaire-Centre National de la Recherche Scientifique, UPR 1086, 1919 Route de Mende, 34293 Montpellier Cedex 05, France



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  		Fig. 1. P63RhoGEF protein. (A) The human protein p63RhoGEF (GenBank accession number AXO02224) and identification by SMART method of the characteristic tandem domain, DH (for Dbl homology domain annotated by SMART as Rho GEF) and PH (pleckstrin homology domain). Segments in purple represent zones of low compositional complexity determined by the SEG program implemented in SMART (Schultz et al., 2000Go). (B) Multiple alignment of human p63RhoGEF DH-PH domains with other well-characterized human GEFs. Stars, double and single points indicate conserved residues, conservative substitutions and similar residues, respectively. Numbering indicates positions of start and end residues for each fragment of sequences used in the alignment. The DH domain is indicated by a solid line, while the PH domain is indicated by a dotted line. The two GEF domains of the protein Trio are displayed as h_trio1 and h_trio2 (AAC34245) (Debant et al., 1996Go). h_dbl (CAA21955; C. Bird, direct submission). h_tiam1 (NP_003244) (Habets et al., 1995Go). h_pix (NP_003890) (Nagase et al., 1995Go). h_duet (AB011422) (Kawai et al., 1999Go). h_vav (P15498) (Katzav et al., 1989Go). h_sos (Q07889) (Chardin et al., 1993Go). (C) Sequence identity dendrogram of DH-PH tandem domains obtained with the evolutionary trace method (Lichtarge et al., 1996Go).

 


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  		Fig. 2. P63RhoGEF mRNA distribution in human tissues. Human multiple tissue northern blot (Clontech) containing approximately 2 µg of poly(A)+ RNA per lane from eight different human tissues was sequentially hybridized with 32P-labeled full-length p63RhoGEF and ß-actin (control for the amount of RNA loaded) cDNA probes. Membranes were exposed for 16 hours (p63RhoGEF) and for 6 hours (ß-actin). The arrow indicates the size of the p63RhoGEF transcript.

 


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  		Fig. 3. P63RhoGEF specifically displays in vitro exchange activity towards RhoA. (A) [3H]GDP nucleotide release assays on different recombinant Rho GTPases (0.4 µM) in absence (control) or in presence of GST-p63RhoGEF DH domain (2.8 µM), or GST-Dbl (1.2 µM). The exchange activity is expressed as the [3H]GDP remaining on the GTPases after 15 minutes of reaction. The experiment presented here is representative of at least three independent assays. Means and standard deviations are shown. (B) [3H]GDP nucleotide release assays on RhoA (0.4 µM) in absence (control) or in presence of GST-Dbl (1.2 µM), GST-p63RhoGEF DH (2.8 µM), or GST-p63RhoGEF L301E (2.8 µM). The exchange activity is represented as in A. (C) Time course study of guanine nucleotide exchange activity on the RhoA GTPase (0.4 µM) in absence ({blacksquare}) or in presence ([UNK]) of GST-p63RhoGEF DH (4 µM). The exchange activity is expressed as in A. (D) Kinetic of association of [35S]GTP{gamma}S to GDP-loaded RhoA (1 µM) in absence ({blacksquare}) or in presence ([UNK]) of p63RhoGEF DH (10 µM). Means and standard deviations are shown.

 


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  		Fig. 4. P63RhoGEF induces RhoA-mediated stress fiber formation. (A) REF-52 cells were transfected with the different constructs as indicated and, 15 hours later, cells were fixed and permeabilized as described in Materials and Methods. The expression of p63RhoGEF was detected by an anti-His monoclonal antibody followed by FITC-coupled anti-mouse immunoglobulins, whereas F-actin was detected by rhodamine-conjugated phalloidin. Expression of GFP-RhoAV14 was visualized directly. When indicated, purified C3 exoenzyme was added in cell medium (0.5 µg/ml) 15 hours before fixation. This experiment is representative of at least three independent experiments, and 100 cells were examined each time. Transfected cells are indicated by white arrows. (B) Quantification of enhancement of stress fiber formation in REF-52 cells transfected as indicated.

 


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  		Fig. 5. P63RhoGEF induces RhoA-mediated stress fiber formation in cardiac myoblasts. After 48 hours of transfection, H9C2 cells expressing His-tagged p63RhoGEF (A) or GFP-RhoAV14 (B) were directly fixed and permeabilized as described in Materials and Methods. The expression of p63RhoGEF was detected by the anti-His antibody followed by FITC-coupled anti-mouse immunoglobulins, whereas F-actin was detected by rhodamine-conjugated phalloidin. This experiment is representative of at least three independent experiments.

 


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  		Fig. 6. Detection of p63RhoGEF mRNA by in situ hybridization in human heart and brain tissue sections. (A) Light microscopy of p63RhoGEF in situ hybridization in human brain. Brightfield (a,e) and darkfield (b) illuminations show the specific hybridization signal observed in body cells localized in the cerebral cortex. Control serial sections were incubated with the sense probe (c,d,f). Bars, 20 µm. (B) Light microscopy of p63RhoGEF in situ hybridization in human heart left ventricle. Brightfield (a,e) and darkfield (b) illuminations show the weak but specific hybridization signal observed in cardiomyocytes. Control sections were incubated with the sense probe (c,d,f). Bar, 10 µm.

 


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  		Fig. 7. Immunolocalization of p63RhoGEF in human heart and brain tissue sections. (A) Detection of p63RhoGEF protein in human brain sections. (a) Normal human cerebellum section using non-specific rabbit IgG at x40 magnification. (b) Normal human cerebellum section at x40 magnification stained with Ab699 antiserum. (c) Enlarged and focused view of the normal human cerebellum section at x100 magnification stained with Ab699 antiserum. The arrow shows one Purkinje cell surrounded by numerous round shaped cerebellar granular cells. (B) Detection of p63RhoGEF protein in human heart sections. (a) Transversal section of normal human heart at x40 magnification stained with Ab699 antiserum. The asterisk indicates the location of a blood vessel. (b) Longitudinal section of normal human heart at x40 magnification stained with Ab699 antiserum. (c) Normal human heart section using rabbit IgG at x40 magnification. (d) Normal human heart section at x40 magnification stained with Ab699 antiserum pre-incubated with the peptide 699 used to generate the anti-p63RhoGEF rabbit polyclonal antibody. (e) Longitudinal cardiac fibers from (b) shown at x100 magnification stained with Ab699 antiserum. (f) Longitudinal cardiac fibers shown at x100 magnification stained with ß-MHC-specific monoclonal antibody. (C) Double immunostaining using rabbit p63RhoGEF polyclonal antibody and either mouse anti-ß-MHC (a,b,c) or mouse anti-vinculin (d,e,f) in human left ventricle. The tissue sections were then analyzed by confocal microscopy. (a,d) p63RhoGEF exhibits a striated pattern of immunolabelling. Arrows in panel a indicate labelling organized in doublets. (b) ß-MHC immunolabelling is found in transverse A-bands (marked as `a') on both sides of the M-line (marked as `m'), which is devoid of labelling. (c) Double immunostaining analysis reveals that the immunolabelled p63RhoGEF doublets are intercalated between A-bands (potentially in the I-band; marked as `i'). (e) Vinculin immunolabelling is found in Z-disks (marked as `z') and at the level of the intercalated disk (*). (f) Double immunostaining analysis reveals that vinculin signal is localized between immunolabelled p63RhoGEF doublet (see arrows). Bars, 10 µm.

 

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