![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 5, 2236-2242, May 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Biological Sciences and the Walther Cancer Institute, University of Notre Dame, Notre Dame, IN 46556;
Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universitat Freiburg, D-79104 Freiburg, Germany; and
Membrane and Cytoskeleton Dynamics Group, Institute Curie, 75248 Paris, France
Submitted August 18, 2005;
Revised February 6, 2006;
Accepted February 8, 2006
Monitoring Editor: Asma Nusrat
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). The intercellular adherens junctions are specialized subapical structures that function as principle mediators of epithelial cell–cell adhesion. The E-cadherin–catenin complex of the adherens junctions effectively links the actin cytoskeleton of adjacent cells to form the subapical adhesion belt of epithelial tissues. Epithelial cell scattering involves the breakdown of adherens junctions, the concomitant loss of the epithelial phenotype and the acquisition of a motile phenotype. The loss of cell–cell contacts during these "epithelial to mesenchymal transitions" has largely been attributed to mutations in components of the adherens junctions or their transcriptional repression (Perez-Moreno et al., 2003). As migrating epithelia do not always exhibit changes in the composition of cadherin–catenin complexes, it is likely that posttranscriptional regulation of cellular processes can impinge on the assembly/disassembly of adherens junctions and contributes to the loss of cell polarity and the acquisition of migratory potential.
Several posttranscriptional mechanisms are implicated in the destabilization of epithelial cell–cell adhesion (D'Souza-Schorey, 2005). Although the phosphorylation of the cadherin–catenin complex has been reported to weaken the interactions between these molecules (Shibamoto et al., 1994; Kinch et al., 1995), the endocytosis of the E-cadherin has also been shown to facilitate the disassembly of cell–cell contacts (Palacios et al., 2001, 2002; Paterson et al., 2003). In addition, control of actin polymerization at sites of cell–cell contact represents a powerful mechanism for regulating the strength of cell–cell adhesion (Vasioukhin et al., 2000; Kovacs et al., 2002). An accumulation of polymerized actin at the adherens junctions stabilizes cell–cell contacts and its disruption leads to the disassembly of the adherens junctions. Epithelial cell scattering has also been shown to require a step mediated by the proteasome (Tsukamoto and Nigam, 1999). The latter study showed that treatment of cells with proteasome inhibitors blocked epithelial cell scattering, although the mechanism by which this occurs is not known. Here we show that Rac1-GTP, a well-known regulator of actin dynamics that localizes to cell junctions, is subject to ubiquitin/proteasome-mediated degradation during the early steps of cell scattering. Deactivation of Rac1 by the ubiquitin/proteasome system (UPS) could represent a decisive mechanism to turn off Rac1-GTP effects and its sustained activation at the adherens junctions.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
(a kind gift from Dr. G. Gacon, Institute Cochin, INSERM, Paris, France) and subcloned into the EcoRI and BamHI sites of pEGFP-C1 (Clontech, Mountain View, CA). Plasmids encoding constitutively active Rac1 (Rac1-G12V pCGT) and dominant negative Rac1 (Rac1-T17N pCGT) were kindly provided by Linda Van Aelst (Cold Spring Harbor, NY) and that encoding POSH-RBD, was provided by Gudila Schmidt (Albert Ludwigs Universitat Freiburg, Germany). Madin Darby canine kidney II (MDCK II) cells and BSC-1 African Green Monkey kidney cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Transient transfections were performed using 8 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) and 6 µg of purified plasmid DNA following manufacturer's instructions. Lactacystin, a proteasome inhibitor (Calbiochem, San Diego, CA), ALLN (Calbiochem) and hepatocyte growth factor/scatter factor (HGF; Calbiochem) were used at a final concentration of 10 µM, 30 µM, and 50 ng/ml, respectively.
|
). Cells were visualized using a Nikon fluorescence microscope (Melville, NY) and a Bio-Rad confocal scanning imaging system (Richmond, CA). Antibodies against the adherens junction components were obtained from Transduction Laboratories (San Diego, CA). Rhodamine phalloidin was obtained from Molecular Probes (Eugene, OR).
Immunoprecipitations and Western Blotting
MDCK II cells grown in 60-mm culture dishes were incubated with 500 µl RIPA lysis buffer (150 mM sodium chloride, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 7.5, and 1% mammalian protease inhibitor cocktail; Sigma, St. Louis, MO) for 4 min on ice with gentle rocking. Cells were scraped and the lysates were centrifuged at 14,000 rpm for 5 min. Cell lysates were incubated with an anti-ubiquitin mouse monoclonal antibody (mAb; Covance, Richmond, CA) for 1 h on ice. Fifty percent slurry of agarose-linked protein A beads (Amersham Biosciences, Piscataway, NJ) was added to the lysate, which was incubated for 1 h at 4°C with rocking. The beads were collected by centrifugation followed by three washes with RIPA lysis buffer. After the final wash the supernatant was discarded and 50 µl of 2x SDS Laemmli buffer was added to the beads. The samples were then resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and probed for Rac1 using mouse mAb against Rac1 (Transduction Laboratories). In a separate set of experiments, MDCK II cells were transiently transfected with plasmids encoding Rac1-G12V pCGT and Rac1-T17N pCGT. Exogenous Rac1 mutants were immunoprecipitated from cell lysates as described above using a mouse mAb against the T7 tag (Novagen, Madison, WI). Immunoprecipitates were probed for ubiquitin with anti-ubiquitin antibodies. In separate experiments, total lysates were probed for ERK and phospho-ERK using anti-ERK and anti-phospho-ERK and (Cell Signaling Technology, Beverly, MA) or for the His-tag using with anti-His antibodies (Upstate Biotechnology, Lake Placid, NY).
PAK Pulldown Assay
Cells were grown in 60-mm dishes and treated with lactacystin and/or HGF for indicated times. Cells were quickly rinsed in phosphate-buffered saline and lysed using 500 µl lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2) plus mammalian protease inhibitors. Cells were scraped, lysates were centrifuged for 5 min at 14,000 rpm. To determine the levels of Rac1-GTP, 400 µl of each cell lysate was incubated with PAK (CRIB)-GST beads for 1 h with rocking at 4°C. Samples were centrifuged for 2 min at 2000 rpm and supernatant was discarded. The beads were washed three times with wash buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2) plus mammalian protease inhibitors. Finally, 50 µl 2x Laemmli buffer was added to the beads and boiled for 10 min. The levels of active Rac1, Rac1-GTP, were detected by Western blotting using specific Rac1 mAb (Transduction Laboratories). Approximately 25 µg of total lysate was analyzed to examine the levels of total Rac1 and -tubulin (Sigma).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). The study showed that inhibitors of proteasome activity such as lactacystin and MG132 blocked cell scattering induced either by v(viral)-Src expression or treatment of cells with HGF/scatter factor. Consistent with these observations, we found that treatment of the MDCK cells with proteasome inhibitors such as lactacystin and ALLN (N-acetyl-Leu-Leu-Nle-CHO, also known as MG101 or LLNL) completely blocked HGF-induced cell scattering. In the absence of lactacystin, HGF treatment promoted the breakdown of E-cadherin–based cell–cell contacts and the acquisition of a motile phenotype (Figure 1). On the basis of these observations, we hypothesized that proteasome-mediated degradation of a junction-stabilizing component is required for cell scattering to proceed.
|
; Takaishi et al., 1997; Noren et al., 2001). Furthermore, Tiam 1, a nucleotide exchange factor of the Rac1 GTPase, localizes to the adherens junctions and its overexpression has similar effects as activated Rac1 on intercellular adhesion (Hordijk et al., 1997). Analysis of Rac1 activation during the sequential steps of cell scattering in MDCK I cells revealed that the Rac1-GTP levels in polarized epithelia are relatively high but that the disassembly of adherens junctions is accompanied by a transient decrease in the cellular levels of Rac1-GTP (Palacios and D'Souza-Schorey, 2003). This is followed by the restoration of high Rac1-GTP as cells become migratory and exhibit lamellipodia and membrane ruffles. The dip in Rac1-GTP levels correlated with a loss of polymerized actin at sites of cell–cell contact (Palacios and D'Souza-Schorey, 2003) and therefore represents potentially a key step in the diminution of adherens junction stability. On the basis of these observations, we investigated whether Rac1-GTP might be subjected to proteasome-mediated degradation during cell scattering. We first examined whether blocking proteasome function reverses the decrease in Rac1-GTP. For these investigations, MDCK II cells were treated with HGF in the presence or absence of lactacystin for increasing time periods and active GTP-bound Rac1 was precipitated from cell lysates using the PAK pulldown assay as previously described (Benard and Bokoch, 2002). As seen in Figure 2A, the level of Rac1-GTP was transiently decreased at 2 and 4 h posttreatment with HGF. This dip in Rac1-GTP levels was also reflected in the cellular levels of total Rac1. However, in the presence of lactacystin, the transient decrease in Rac1-GTP (and total Rac1) was not observed (Figure 2A). This finding indicated that proteasome activity has a role in the down-regulation of Rac1-GTP during cell scattering. In contrast to Rac1, RhoA levels remained unperturbed during cell scattering in the presence or absence of lactacystin (Supplementary Figure 1). The decline in Rac1-GTP levels was not restricted to HGF treatment only, because EGF treatment also induced a transient decrease in Rac1-GTP levels (Supplementary Figure 2). A similar effect on Rac1-GTP levels during cell scattering was also observed in other epithelial cell lines, such as BSC-1 African Green monkey kidney cells (Figure 2B). It should be noted however, that although BSC-1 cells exhibited the decline in Rac1-GTP levels, the "total" Rac1 levels remained constant. This profile for total Rac1 is consistent with that previously described in the MDCK I cell line (Palacios et al., 2003). One explanation for this is that Rac1-GAP activity may be significantly higher in the MDCK I and BSC I cell lines during the early stages of cell scattering. This in turn leads to a substantial pool of Rac1-GDP that is less susceptible to proteasome degradation (see below). Nevertheless, Rac1-GTP, which is responsible for the maintenance of cell–cell contacts, is subject to proteasome-mediated degradation during cell scattering, and this appears to be essential for cell scattering, irrespective of the cell type. It should also be noted that the down-regulation of Rac1-GTP is likely dependent on growth factor–mediated signaling because physical disruption of cell–cell contacts by depletion of extracellular calcium did not produce a down-regulation of Rac1-GTP levels (unpublished data). The effectiveness of lactacystin treatment was tested by analyzing lysates of cells treated with proteasome inhibitors on SDS gels. These studies showed smears of ubiquitinated proteins in lactacystin-treated cells, suggesting an increase in the cellular levels of ubiquitinated proteins upon proteasome inhibition (Supplementary Figure 3). We also determined whether the effect of proteasome inhibition was specific to growth factor–induced Rac1 degradation or whether other cellular processes activated by HGF during cell scattering were also perturbed. To this end, we analyzed the levels of ERK (extracellular signal regulated kinase) activation in response to HGF. As shown in Figure 3, HGF-induced phosphorylation and activation of ERK was not altered in the presence of proteasome inhibitors. No changes were detected even at more prolonged time periods (unpublished data). In addition, tyrosine phosphorylation of E-cadherin, which also occurs in response to HGF, was not perturbed by proteasome inhibition (unpublished data).
|
). Thus, we examined whether a nondegradable isoform of Rac will protect against HGF-induced cell scattering by substituting for Rac1. Thus, MDCK cells transiently expressing Rac2-GFP were treated with HGF under conditions where nontransfected cells exhibited a scattered phenotype (Figure 4A). We found that almost all Rac2-GFP-expressing cells remained adhered to adjoining cells and actin staining between cells was stabilized. In contrast, untransfected cells, or cells expressing GFP alone, detached from the cell colony. Representative images are shown (Figure 4A). We also attempted to examine the effects of the Rac1 mutants [Rac1(K147R), Rac1(R174A,L177A)] resistant to proteasome degradation. These mutants did not dominantly inhibit degradation of endogenous Rac1 (unpublished data), but we could not examine their effects on cell scattering due to low transfection efficiency.
|
Previous studies have indicated that effector interaction is necessary for Rac1 degradation (Pop et al., 2004). As such, overexpression of a specific GTPase-binding domain of the Rac-specific effector, POSH (POSH-RBD), prevented Rac1-GTP degradation in HEK293 cells treated with CNF1. Thus we transfected MDCK II cells with a plasmid expressing His–tagged POSH-RBD and then induced them to scatter by treatment with HGF. As shown in Figure 4B, endogenous Rac1-GTP degradation was clearly inhibited in POSH-RBD–expressing cells. Furthermore, the percentage of cells that scattered was significantly decreased (Figure 4C). These data suggest that blocking the interaction of Rac1-GTP with its downstream effector by overexpressing POSH-RBD prevents degradation of the GTPase and attests to the importance of functional Rac1 levels during cell scattering.
Next, we examined whether Rac1-GTP itself might be a substrate of the proteasome during cell scattering. Thus, we tested whether Rac1 was modified by ubiquitin-tagging during the early steps of cell scattering. To this end, ubiquitin-tagged proteins in HGF-treated cell lysates were analyzed specifically for ubiquitinated Rac1 by immunoprecipitation with a mAb directed against ubiquitin, followed by probing of the immunoprecipitates for Rac1 using Western blotting procedures. As seen in Figure 5, a significant amount of ubiquitin-tagged Rac1 was observed at 2 h after HGF treatment, which then subsequently declined likely because of its degradation by the proteasome. In contrast, in the presence of the proteasome inhibitor, ubiquitinated Rac1 was detected in cell lysates even at prolonged periods after HGF treatment. The sizes of ubiquitin-tagged Rac1 bands were approximately 34 and 40 kDa, consistent with short-chain polyubiquitination. These modified higher molecular weight forms of Rac1 were not observed in Rac1-GTP pulldown assays, which may be because ubiquitin-tagged Rac1 is not efficiently recognized by PAK. A very low level of ubiquitinated Rac1 was also detected in polarized cells before scattering, suggesting that a low basal level of Rac1-GTP degradation might occur even under normal conditions. When the anti-ubiquitin antibody was replaced with a nonrelevant control antibody in the experiments described above, and then probed for Rac1 by Western blotting, ubiquitin-tagged Rac1 was not observed in the immunoprecipitate (Supplementary Figure 4).
|
We also examined whether Rac1-GTP rather than Rac1-GDP was specifically modified with ubiquitin and targeted to the proteasome for degradation. MDCK II cells were transfected with mammalian expression plasmids encoding constitutively active Rac1, Rac1-G12V, or dominant negative Rac1, Rac1-T17N, that were tagged with the viral T7 epitope. Exogenous Rac1-GTP and Rac1-GDP were immunoprecipitated using an anti-T7 mAb. Immunoprecipitates were resolved on SDS gels and labeled for ubiquitin using Western blotting techniques. As seen in Figure 6, the Rac1-GTP mutant, Rac1-G12V is a significantly better substrate for ubiquitination compared with the GDP-bound mutant, Rac1-T17N, and although the expression of Rac1-T17N is higher than Rac1-G12V, minimal labeling of Rac1-T17N with ubiquitin was observed. Thus, Rac1-GTP is selectively targeted to the UPS system for its deactivation during epithelial cell scattering. These results are consistent with previous findings showing nucleotide specific degradation of Rac1 by the proteasome (Doye et al., 2002, Pop et al., 2004).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; Lerm et al., 2002; Wang et al., 2003). Ubiquitin tagging of Rac1-GTP during scattering may involve the activation of a ubiquitin ligase or the modification of the Rac1 GTPase itself to render it a better substrate for ubiquitin tagging. It is also possible that additional proteins besides Rac1 may also be subject to proteasome-mediated degradation to facilitate the destabilization of the adherens junctions. In this regard, -catenin contains a PEST sequence making it a potential target for proteasome-mediated degradation (Takahashi et al., 2000). The cytoplasmic domain of E-cadherin also contains a PEST sequence although the latter is masked by 
-catenin and p120 catenin (Huber et al., 2001). However, analyses of protein expression by Western blotting (Supplementary Figure 5) and immunofluorescence microscopy (unpublished data), using antibodies directed against -catenin, -catenin, p120 catenin, and E-cadherin, revealed that the adherens junction components were not degraded at early stages of cell scattering, i.e., at 2 h after treatment with HGF. However, we cannot exclude that other proteins in addition to Rac1-GTP might also be degraded during the dissolution of cell–cell contacts. Identification of the ubiquitin ligase that is responsible for Rac1 modification during adherens junction disassembly will shed significant light in this regard.
The deactivation of Rac1 as described above, could serve to promote the destabilization of the actin cytoskeleton at the subapical adhesion belt of epithelial monolayers. This in turn might "loosen up" the adherens junctions to facilitate processes such as the endocytosis of E-cadherin. It would seem more efficient if adherens junctions disassembly is accompanied by the dissolution of actin bundles in order to facilitate membrane internalization at sites of cell–cell contact. What might cause the transient deactivation of Rac1 to promote cytoskeletal destabilization at the adherens junctions? Although the ubiquitination and proteasome-mediated degradation of Rac1 might serve as the initial step to "set off" the disassembly of adherens junctions, it is likely that other downstream mechanisms also contribute toward the down-regulation of Rac1 to bring about the disassembly of epithelial cell–cell contacts. Although, little is know about Rac1-GAP activities during the disassembly of cell–cell contacts it is possible that GAP activities might also serve to decrease the Rac1-GTP pool. Furthermore, previous work in our laboratory has shown that activation of the ARF6 GTPase promotes the decrease in Rac1-GTP levels in addition to facilitating the endocytosis of E-cadherin. These effects of ARF6 are mediated by the recruitment of nm23-H1, a nucleoside diphosphate kinase, shown to function as a GTP-source for dynamin to facilitate endocytosis (Krishnan et al., 2001), as well as to down-regulate cellular levels of Rac1-GTP by binding and sequestering Tiam-1 (Otsuki et al., 2001). Thus several cellular mechanisms exist to bring about the depletion of Rac1-GTP pools and subsequently actin cytoskeleton disassembly, during the breakdown of adherens junctions. The relative activity of each of these processes in different cell types is an area for future investigation.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Crislyn D'Souza-Schorey (D'Souza-Schorey.1{at}nd.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Braga, V. M., Machesky, L. M., Hall, A., and Hotchin, N. A. ((1997). ). The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol. 137, , 1421–1431.
Doye, A., Mettouchi, A., Bossis, G., Clement, R., Buisson-Touati, C., Flatau, G., Gagnoux, L., Piechaczyk, M., Boquet, P., and Lemichez, E. ((2002). ). CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111, , 553–564.[CrossRef][Medline]
D'Souza-Schorey, C. ((2005). ). Disassembling adherens junctions: breaking up is hard to do. Trends Cell Biol. 15, , 19–26.[CrossRef][Medline]
Hordijk, P. L., ten Klooster, J. P., van der Kammen, R. A., Michiels, F., Oomen, L. C., and Collard, J. G. ((1997). ). Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science 278, , 1464–1466.
Huber, A. H., Stewart, D. B., Laurents, D. V., Nelson, W. J., and Weis, W. I. ((2001). ). The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover. J. Biol. Chem. 276, , 12301–12309.
Kinch, M. S., Clark, G. J., Der, C. J., and Burridge, K. ((1995). ). Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia. J. Cell Biol. 130, , 461–471.
Kovacs, E. M., Goodwin, M., Ali, R. G., Paterson, A. D., and Yap, A. S. ((2002). ). Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr. Biol. 12, , 379–382.[CrossRef][Medline]
Krishnan, K. S., Rikhy, R., Rao, S., Shivalkar, M., Mosko, M., Narayanan, R., Etter, P., Estes, P. S., and Ramaswami, M. ((2001). ). Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling. Neuron 30, , 197–210.[CrossRef][Medline]
Lerm, M., Pop, M., Fritz, G., Aktories, K., and Schmidt, G. ((2002). ). Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac. Infect. Immun. 70, , 4053–4058.
Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. ((2001). ). Cadherin engagement regulates Rho family GTPases. J. Biol. Chem. 276, , 33305–33308.
Otsuki, Y., Tanaka, M., Yoshii, S., Kawazoe, N., Nakaya, K., and Sugimura, H. ((2001). ). Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1. Proc. Natl. Acad. Sci. USA 98, , 4385–4390.
Palacios, F., and D'Souza-Schorey, C. ((2003). ). Modulation of Rac1 and ARF6 activation during epithelial cell scattering. J. Biol. Chem. 278, , 17395–17400.
Palacios, F., Price, L., Schweitzer, J., Collard, J. G., and D'Souza-Schorey, C. ((2001). ). An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration. EMBO J. 20, , 4973–4986.[CrossRef][Medline]
Palacios, F., Schweitzer, J. K., Boshans, R. L., and D'Souza-Schorey, C. ((2002). ). ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly. Nat. Cell Biol. 4, , 929–936.[CrossRef][Medline]
Paterson, A. D., Parton, R. G., Ferguson, C., Stow, J. L., and Yap, A. S. ((2003). ). Characterization of E-cadherin endocytosis in isolated MCF-7 and chinese hamster ovary cells: the initial fate of unbound E-cadherin. J. Biol. Chem. 278, , 21050–21057.
Perez-Moreno, M., Jamora, C., and Fuchs, E. ((2003). ). Sticky business: orchestrating cellular signals at adherens junctions. Cell 112, , 535–548.[CrossRef][Medline]
Pop, M., Aktories, K., and Schmidt, G. ((2004). ). Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1. J. Biol. Chem. 279, , 35840–35848.
Shibamoto, S., Hayakawa, M., Takeuchi, K., Hori, T., Oku, N., Miyazawa, K., Kitamura, N., Takeichi, M., and Ito, F. ((1994). ). Tyrosine phosphorylation of beta-catenin and plakoglobin enhanced by hepatocyte growth factor and epidermal growth factor in human carcinoma cells. Cell Adhes. Commun. 1, , 295–305.[Medline]
Takahashi, N., Ishihara, S., Takada, S., Tsukita, S., and Nagafuchi, A. ((2000). ). Posttranscriptional regulation of alpha-catenin expression is required for Wnt signaling in L cells. Biochem. Biophys. Res. Commun. 277, , 691–698.[CrossRef][Medline]
Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H., and Takai, Y. ((1997). ). Regulation of cell-cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139, , 1047–1059.
Tsukamoto, T., and Nigam, S. K. ((1999). ). Cell-cell dissociation upon epithelial cell scattering requires a step mediated by the proteasome. J. Biol. Chem. 274, , 24579–24584.
Vasioukhin, V., Bauer, C., Yin, M., and Fuchs, E. ((2000). ). Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100, , 209–219.[CrossRef][Medline]
Wang, H. R., Zhang, Y., Ozdamar, B., Ogunjimi, A. A., Alexandrova, E., Thomsen, G. H., and Wrana, J. L. ((2003). ). Regulation of cell polarity and protrusion formation by targeting RhoA for degradation. Science 302, , 1775–1779.
This article has been cited by other articles:
|
|
 |
|
  S. Busche, A. Descot, S. Julien, H. Genth, and G. Posern Epithelial cell-cell contacts regulate SRF-mediated transcription via Rac-actin-MAL signalling J. Cell Sci., April 1, 2008; 121(7): 1025 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yano, I. Kobayashi, Y. Onodera, F. Luton, M. Franco, Y. Mazaki, S. Hashimoto, K. Iwai, Z. Ronai, and H. Sabe Fbx8 Makes Arf6 Refractory to Function via Ubiquitination Mol. Biol. Cell, March 1, 2008; 19(3): 822 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Yi, E. Nagyova, G. Manandhar, R. Prochazka, M. Sutovsky, C.-S. Park, and P. Sutovsky Proteolytic Activity of the 26S Proteasome Is Required for the Meiotic Resumption, Germinal Vesicle Breakdown, and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Matured In Vitro Biol Reprod, January 1, 2008; 78(1): 115 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. H. Psahoulia, S. Moumtzi, M. L. Roberts, T. Sasazuki, S. Shirasawa, and A. Pintzas Quercetin mediates preferential degradation of oncogenic Ras and causes autophagy in Ha-RAS-transformed human colon cells Carcinogenesis, May 1, 2007; 28(5): 1021 - 1031. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
38 and 44 kDa. HC, IgG heavy chain, LC, IgG light chain, Ub, ubiquitin.