CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells - PubMed (original) (raw)

. 2006 Jun;17(6):2489-97.

doi: 10.1091/mbc.e05-09-0876. Epub 2006 Mar 15.

Laurent Turchi, Benoit Desnues, Anne Doye, Gilles Ponzio, Jean-Louis Mege, Motozo Yamashita, Ying E Zhang, Jacques Bertoglio, Gilles Flatau, Patrice Boquet, Emmanuel Lemichez

Affiliations

CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells

Laurent Boyer et al. Mol Biol Cell. 2006 Jun.

Abstract

Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Depletion of Smurf1 impairs the CNF1-induced cellular depletion of RhoA. (A) Immunoblot anti-FLAG. Smurf1-RNAi expression impairs the ectopic expression of Smurf1. HEK293 were cotransfected using calcium phosphate with 200 ng of FLAG-Smurf1 expression plasmid and different doses of expression plasmids of either Smurf1-RNAi or control-RNAi. Cells were lysed 24 h after transfection. (B) Immunoblots showing the kinetics of CNF1-induced RhoA depletion in HEK293 cells transfected with either Smurf1-RNAi or control-RNAi expression plasmids. Cells were transfected 24 h before addition of 10−9 M CNF1. Equal protein loading was verified by anti-actin immunoblotting. (C) Immunoblots showing the kinetics of CNF1-induced RhoA and Rac depletion in either Wild-type or _Smurf1_-deficient MEF cells intoxicated with 10−9 M CNF1. Equal protein loading was verified by anti-actin immunoblotting. (D) Quantification of the CNF1-induced RhoA and Rac depletion (mean values of 2 independent experiments). (E) Immunoblots showing the kinetic of CNF1-induced activation of RhoA in both wild-type and _Smurf1_-deficient MEF cells. Cells were treated with 10−9 M CNF1 and processed for GST-Rhotekin pull-down (labeled RhoAGTP). Total RhoA protein used in the assay was analyzed on 2% of the total cell lysates (labeled RhoA). Equal amounts of protein used in the assays were verified by anti-actin immunoblotting. (F) Quantification of CNF1-induced activation of Rho in wild-type and _Smurf1_-deficient MEF cells (mean values of 2 independent experiments).

Figure 2.

Figure 2.

Relationship between Smurf1 and actin reorganization induced by CNF1. (A–D) Cells were fixed and processed for actin cytoskeleton labeling using tetramethylrhodamine B isothiocyanate-phalloidin. Bars, 10 μm. (A) Effect of Smurf1-RNAi on actin cytoskeleton reorganization induced by CNF1 in HEK293 cells. Cells were cotranfected with Smurf1-RNAi expression plasmid together with pEGFP-C2. Twenty-four hours after transfection cells were intoxicated for another 24 h with 10−9 M CNF1. This picture shows a transfected cell, which displays actin cables in contrast to nontransfected cells. (B and C) Reorganization of the actin cytoskeleton in either wild-type (B) or _Smurf1_-deficient (C) MEF cells intoxicated 4 or 24 h by 10−9 M CNF1. (D) Ectopic expression of Smurf1, at the difference with Smurf1-C699A, impairs the formation of actin cables induced by 24 h of intoxication of _Smurf1_-deficient MEF cells by 10−9 M CNF1. _Smurf1_-deficient MEF cells were transfected with pEYFP-Flag-Smurf1 or pEYFP-Flag-Smurf1C699A expression plasmids and intoxicated 16 h after transfection. Inset pictures show the transfected cells.

Figure 3.

Figure 3.

Specificity of actin cytoskeleton reorganization induced by CNF1 in cell lines. (A and D) Cells were fixed and processed for actin cytoskeleton labeling using TRITC-phalloidin. Bars, 10 μm. (A) Ectopic expression of Smurf1 impairs the formation of actin cables induced by 24 h of Vero cell intoxication by 10−9 M CNF1. Vero cells were transfected with pEYFP-Flag-Smurf1 expression plasmid and intoxicated 16 h after transfection. Inset picture shows the transfected cell. (B) Immunoblot anti-Smurf1. Cell protein extracts (100 μg) were resolved on 7% SDS-PAGE before anti-Smurf1 immunoblotting (H-60 used at 1/400). (C) Histogram showing the relative expression level of Smurf1 mRNA in cell lines and HUVECs determined by real-time quantitative PCR (mean values of 2 independent experiments). Total RNA was prepared from 106 cells using RNeasy kit (QIAGEN). (D) HEp-2, HEK293, and 804G cells were intoxicated with 10−9 M CNF1 for the indicated times.

Figure 4.

Figure 4.

Specificity of CNF1-induced Rho protein depletion in cell lines and primary cell types. (A) Immunoblots showing the kinetics of CNF1-induced Rho, Rac, and Cdc42 depletion in HEp-2, Vero, HEK293, and 804G cell lines. Cells were treated for different times with 10−9 M CNF1. Cells were lysed and immunoblots were performed on 40 μg of the total protein lysate. Immunoblots anti-actin were used to evaluate equal loading and normalize Rho GTPase signals. (B) Quantification of the CNF1-induced depletion of Rho proteins. Cellular depletion of Rho proteins was measured at 24 h of cell intoxication with 10−9 M CNF1 (mean values of 2 independent experiments). (C) Immunoblots showing the kinetics of CNF1-induced Rho, Rac, and Cdc42 depletion in primary HUVECs, macrophages, keratinocytes, and fibroblasts. Cells were treated for different times with 10−9 M CNF1. Cells were lysed and immunoblots were performed using 40 μg of the total protein lysate. Immunoblots anti-actin were used to evaluate equal loading and normalize Rho GTPases signals. (D) Quantification of the CNF1-induced depletion of Rho proteins. Cellular depletion of Rho proteins was measured at 24 h of cell intoxication with 10−9 M CNF1 (mean values of 2 independent experiments).

Figure 5.

Figure 5.

Kinetics of CNF1-induced Rho protein activation. (A) Immunoblots showing the kinetics of activation and degradation of Rho proteins in HEp-2 cells after different times of 10−9 M CNF1 exposure. RhoGTP, RacGTP, and Cdc42GTP indicate the extent of Rho protein activation measured using GST pull-down assays. Bottom, corresponding kinetics of CNF1-induced Rho proteins depletion. (B) Immunoblots showing the CNF1-induced Rho, Rac, and Cdc42 sustained activation in, respectively, HEp-2, Vero, and 293 cells. Activation of Rho proteins was measured using GST pull-down assays.

Figure 6.

Figure 6.

Specificity of permanently activated Rho protein ubiquitylation. (A) Comparison of permanently activated Rho protein cellular ubiquitylation efficiencies. Cells expressing permanently activated HA-tagged Rho proteins and His-Ub were processed for histidine-tag purification after HA-Rho protein levels normalization. His-Ub cross-linked forms of HA-Rho proteins were visualized by immunoblotting anti-HA. An anti-HA immunoblot was performed in parallel on 2% of the total lysates to compare the amounts of total HA-Rho proteins used in the His-Ub purification (labeled total HA-Rac, HA-Rho, and HA-Cdc42). (B) Comparison of permanently activated Rac ubiquitylation efficiencies in four cell lines. Cells expressing permanently activated HA-tagged Rac and His-Ub were processed for histidine-tag purification after HA-Rho protein levels normalization. His-Ub cross-linked forms of HA-Rac were visualized by immunoblotting anti-HA. An anti-HA immunoblot was performed in parallel on 2% of the total lysates to compare the amounts of total HA-Rac used in the His-Ub purification (labeled total HA-Rac).

Figure 7.

Figure 7.

Effect of Smurf1 on permanently activated RhoA ubiquitylation in Vero cells. (A) Immunoblots anti-HA showing RhoA ubiquitylation efficiencies. Vero cells were cotransfected 8 h with expression plasmids of His-Ub, HA-RhoAL63, or wild-type HA-RhoA and pYFP-Flag-Smurf1, as indicated. Cells were processed for histidine-tag purification directly or after 6 h of intoxication with 10−9 M CNF1, as indicated. Equal levels of HA-Rho proteins were engaged in the His-Ub purification assays. His-Ub cross-linked forms of HA-RhoA were visualized by immunoblotting anti-HA. In parallel, an anti-HA immunoblot was performed on 2% of the total lysates to compare the amounts of total HA-RhoA used in the His-Ub purification (labeled total HA-RhoA). (B) Two independent experiments were quantified for RhoA ubiquitylation levels, compared with total RhoA in 2% of the cell lysates.

References

    1. Atfi A., Djelloul S., Chastre E., Davis R., Gespach C. Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor beta-mediated signaling. J. Biol. Chem. 1997;272:1429–1432. - PubMed
    1. Barbieri J. T., Riese M. J., Aktories K. Bacterial toxins that modify the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 2002;18:315–344. - PubMed
    1. Boettner B., Van Aelst L. The role of Rho GTPases in disease development. Gene. 2002;286:155–174. - PubMed
    1. Boquet P., Lemichez E. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis? Trends Cell Biol. 2003;13:238–246. - PubMed
    1. Burridge K., Wennerberg K. Rho and Rac take center stage. Cell. 2004;116:167–179. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources