Report |
Address correspondence to Ahmed Zahraoui, Laboratory of Morphogenesis and Cell Signaling, Centre National de la Recherche Scientifique, UMR144 Institut Curie, 26 rue d'Ulm, 75248, Paris, Cedex 05, France. Tel.: 33-1-42346370. Fax: 33-1-42346377. email: zahraoui{at}curie.fr
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Abstract |
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Key Words: Rab GTPase; phosphorylation; VASP; cellcell junctions; epithelial cells
Abbreviations used in this paper: aPKC
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Introduction |
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Another family of small GTPases, Rab proteins, is also involved in regulating TJ dynamics. Rab GTPases seem to regulate specific membrane transport events including vesicle formation, motility via kinesins or myosins, tethering, and fusion (Pfeffer, 2001; Zerial and McBride, 2001). We have shown previously that Rab13 is recruited from a cytoplasmic pool to cellcell contacts at an early stage during TJ assembly (Zahraoui et al., 1994; Sheth et al., 2000). Expression of the active (Rab13Q67L), but not the inactive (Rab13T22N) mutant of Rab13 in epithelial MDCK cells delays the recruitment of TJ proteins, claudin1 and ZO-1, and alters TJ integrity. Interestingly, it does not impair the recruitment of E-cadherin to the lateral membrane (Marzesco et al., 2002). Nevertheless, how Rab13 interferes with TJ structure and function is still unclear. In the present work, we show that the active mutant Rab13Q67L inhibits PKA-mediated phosphorylation of VASP and its recruitment to cellcell junctions. Moreover, Rab13GTP binds to PKA and inhibits its activity. Our data provide new insights into the molecular mechanisms by which Rab13 regulates TJ assembly in epithelial cells.
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Results and discussion |
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Activation of PKA abolishes the inhibitory effect of Rab13Q67L on the recruitment of TJ proteins, ZO-1 and claudin1
To determine whether the effect of Rab13 on claudin1 and ZO-1 recruitment could be mediated by PKA, we examined the consequences of PKA activation on the recruitment of ZO-1 and claudin1 in cells expressing GFP-Rab13Q67L using the Ca2+ switch assay. In control cells as well as in cells expressing GFP-Rab13T22N or GFP-Rab13Q67L, removal of Ca2+ resulted in the dissociation of cellcell junctions and redistribution of ZO-1 and claudin1 into the cytoplasm (Fig. 4). By 2 h after addition of Ca2+, ZO-1 immunoreactivity was already detected at cellcell junctions in cells expressing GFP and GFP-Rab13T22N. In contrast, expression of GFP-Rab13Q67L delayed slightly the recruitment of ZO-1; ZO-1 labeling was discontinuous along the lateral membrane. By 4 h after readdition of calcium, ZO-1 staining was detected at the plasma membrane, but some ZO-1 immunoreactivity was still observed in the cytoplasm in cells expressing GFP-Rab13Q67L (Fig. 4 A). Similarly, plasma membrane recruitment of claudin1 was delayed in cells expressing GFP-Rab13Q67L. Within 612 h after readdition of calcium, most of the claudin1 staining was still cytoplasmic in GFP-Rab13Q67L cells, whereas claudin1 was detected at the plasma membrane in cells expressing GFP or GFP-Rab13T22N (Fig. 4 B). Significantly, activation of PKA with Forskolin restored the kinetics of both ZO-1 and claudin1 recruitment to the lateral membrane in GFP-Rab13Q67L cells (Fig. 4). This indicates that PKA activation abolished the inhibitory effect of Rab13GTP on the recruitment of TJ proteins, leading to their accumulation at cellcell junctions. Our data establish that the Rab13PKA signaling complex plays a key role in TJ biogenesis. They further confirm that PKA-dependent phosphorylation is implicated in the dynamics of epithelial TJ.
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In this paper, we identified PKA as a new effector for the small GTPase Rab13. Our data demonstrate that the GTP bound form of Rab13 directly binds and negatively controls PKA activity both in vitro and in vivo. This is the first demonstration of a biochemical and functional link between a small Rab GTPase and the PKA catalytic subunit.
Protein phosphorylation/dephosphorylation events are required for the regulation of TJ dynamics (Sakakibara et al., 1997; Matter and Balda, 2003). In these processes, VASP may play a key role due to its differential actin binding capacities depending on its state of phosphorylation. Phosphorylation of VASP by PKA reduces VASPactin interactions and promotes the association of P-VASP with cellcell junctions. Interestingly, VASP is required for the establishment of cellcell contacts (Vasioukhin et al., 2000). Although Rab13 does not affect the recruitment of E-cadherin (Marzesco et al., 2002) and ß-catenin to cellcell junctions (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200312118/DC1), we cannot rule out the possibility that Rab13 alters TJ assembly through regulating the recruitment of other adherens junction components. We suggest that Rab13GTP could locally influence actin rearrangement by controlling PKA-dependent VASP phosphorylation and hence inhibit TJ integrity. Our previous data suggested that Rab13, in its GTP-bound form, interacts with an effector that inhibits the recruitment of claudin1 and, to a lesser extent, that of ZO-1 to TJ. Conversely, the inactive form Rab13T22N, unable to bind the effector, favors the establishment of TJ gate and fence functions (Marzesco et al., 2002). The inhibitory effect of the Rab13Q67L mutant on TJ assembly and function could thus be explained by the inhibition of PKA activity. The Rab13PKA signaling complex may as well regulate vesicle docking near cellcell junctions. Indeed, PKA activity has been shown to be required for membrane trafficking (Pimplikar and Simons, 1994; Rodionov et al., 2003). Therefore, our observations allow us to postulate the existence of a new signaling cascade at the TJ implicating Rab13PKA in the dynamics of TJ assembly. They also highlight novel insights into the role of Rab proteins in membranecytoskeleton interactions.
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Materials and methods |
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Cell culture
MDCK cells (clone II) were cultured in DME supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 10 mg/ml streptomycin. The cells were incubated at 37°C under 10% CO2 atmosphere. MDCK cells stably expressing GFP, GFP-Rab13T22N, or GFP-Rab13Q67L were described previously (Marzesco et al., 2002).
Incubation with kinase inhibitors/activators
500,000 cells were plated onto 3.5-cm diam culture plates overnight and incubated as follows: 1 h at 37°C with 30 µM of PKA inhibitor H-89; 30 min with the PKC and PKG inhibitors Rö-32-0432 (10 µM) or KT5823 (5 µM; Calbiochem); 30 min with 100 µM cAMP or cGMP; or 20 min with 10 µM Forskolin (Sigma-Aldrich). Cells were washed three times in PBS and subjected to either immunofluorescence or immunoblotting procedures. We confirmed that Rö-32-0432 is effective in inhibiting PKC activity under these conditions.
Ca2+ switch experiments
MDCK cells expressing GFP or GFP-Rab13T22N and GFP-Rab13Q67L mutants were plated at a density of 10,000,000 cells/cm2, and incubated for 16 h in S-MEM medium without Ca2+. Cells were rinsed with normal DME, incubated in DME for the indicated time and analyzed by immunofluorescence. When the effect of PKA activation/inhibition was studied, Forskolin and H89 were added to DME after the Ca2+depletion.
Immunofluorescence microscopy
Immunofluorescence was performed as described previously (Marzesco et al., 2002). The samples were analyzed with a fluorescence microscope (Carl Zeiss MicroImaging Inc.) and further processed with Adobe Photoshop Software.
Immunoprecipitation and immunoblotting
500,000 MDCK or cells expressing GFP, GFP-Rab13T22N, or GFP-Rab13T22N were grown on 3.5-cm diam culture plates for 16 h, washed three times with ice-cold PBS, and extracted in 0.5% Triton, 10 mM Tris-HCl, pH 7.6, 120 mM NaCl, 25 mM KCl, 1.8 mM CaCl, 1 mM sodium vanadate, 50 mM NaF, and a mixture of protease inhibitors (Sigma-Aldrich) on a rocker platform for 30 min at 4°C. In the GST pull-down experiments, cells were grown on 10-cm diam culture plates for 3 d, washed in PBS, and extracted in the same buffer containing 1% NP-40 instead of Triton X-100. Solubilized material was recovered by pelleting at 18.000 g for 15 min at 4°C. Supernatants were collected and protein concentration determined using the protein assay kit (Bio-Rad Laboratories). For immunoprecipitation, cells expressing GFP-Rab13Q67L were grown for 3 d on 10-cm diam culture plates, washed with PBS, and extracted in IP buffer (50 mM NaCl, 25 mM Tris, pH 8, 1 mM EDTA, 0.25% Triton, 1 mM sodium vanadate, 50 mM NaF, and protease inhibitors). After centrifugation, supernantants were incubated with 5 µg of anti-VASP antibody over night at 4°C. Protein G agarose beads were added for 2 h and the beads washed three times with IP buffer. Equal amount of protein were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose filters. Filters were probed with anti-VASP or antiP-VASP antibodies before ECL detection according to the manufacturer's protocols (Pierce Chemical Co.).
GST pull-down assay
GST-Rab13 fusion protein was expressed in E. coli and purified according to the manufacturer's protocol (Amersham Biosciences). GST-Rab13 bound to glutathione beads was loaded with 1 mM GDP or GTPS (a poorly hydrolyzable GTP analogue) for 90 min at RT in incubation buffer (100 mM NaCl, 20 mM Tris, 10 mM EDTA, 5 mM MgCl2, and 1 mM DTT, pH 7.6). After washing, beads were incubated overnight with MDCK cell extracts at 4°C. To prove the direct interaction of Rab13 with PKA, 100 µg of purified PKA catalytic subunit from bovine heart (Sigma-Aldrich) was incubated with a 10-fold molar excess of purified GST, GST-Rab13-GDP, or GST-Rab13-GTP
S proteins in the incubation buffer overnight at 4°C. GST-Rab6 (a gift from B. Goud and S. Monier, UMR144 Institut Curie) was used as negative control. After washing, the extent of PKA binding was determined by SDS-PAGE and Western blotting using polyclonal anti-PKA
cat or anti-PKC
antibodies.
In vitro kinase assay
10 U of purified PKA catalytic subunit was incubated with VASP immunoprecipitate and either 1 µg of protein kinase inhibitor peptide, PKI (Sigma-Aldrich), and 10 µg of purified GST-Rab13 loaded with GTPS or GST in kinase buffer (100 mM NaCl, 20 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 mM ATP) for 30 min at 30°C. The reaction was stopped by adding 3x SDS sample buffer, separated on SDS-PAGE, and VASP phosphorylation determined by Western blot using the anti-VASP antibody.
Online supplemental material
The online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312118/DC1. Fig. S1 shows that Rab13 mutants do neither alter the recruitment of adherens junction proteins such as (A) ß-catenin or (B) afadin nor (C) cortical actin in a calcium switch experiment. Antiß-catenin and anti-afadin antibodies were purchased from Sigma-Aldrich and BD Transduction Laboratories, respectively. Phalloidin was purchased from Sigma-Aldrich. Supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.jcb200312118/DC1.
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Acknowledgments |
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This work was supported by grants from Centre National de la Recherche Scientifique, Institut Curie, and the Association pour la Recherche sur le Cancer (ARC 3457) to A. Zahraoui. K. Köhler is a recipient of a fellowship from the Swiss National Science Foundation.
Submitted: 16 December 2003
Accepted: 19 March 2004
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