Marc Piechaczyk

CNF1 Exploits the Ubiquitin-Proteasome Machinery to Restrict Rho GTPase Activation for Bacterial Host Cell Invasion

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.

The HBZ Factor of Human T-cell Leukemia Virus Type I Dimerizes with Transcription Factors JunB and c-Jun and Modulates Their Transcriptional Activity

The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-κB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813–12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax.

Ubiquitin-independent degradation of proteins by the proteasome.

The proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here.

Proteolysis by calpains: a possible contribution to degradation of p53.

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals

We report here that transport of the protein product of the c-fos proto-oncogene from the cytoplasm, where it is synthesized, into the nucleus, where it operates as part of the AP-1 transcription complex, is not spontaneous but depends on the continuous stimulation of cells by serum factors. A labile protein inhibitor of transport, the effect of which is reversed by cAMP, is responsible for retention of c-Fos protein within the cytoplasm of serum-starved fibroblasts. In contrast, v-Fos proteins transduced by the murine retroviruses FBJ and FBR, which remain nuclear in the absence of serum, evade the translocation control, which therefore appears to contribute to their tumorigenic potential.

Down-Regulation of c-Fos/c-Jun AP-1 Dimer Activity by Sumoylation

ABSTRACT The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

SUMO under stress.

During the last decade, SUMOylation has emerged as a central regulatory post-translational modification in the control of the fate and function of proteins. However, how SUMOylation is regulated itself has just started to be delineated. It appears now that SUMO (small ubiquitin-related modifier) conjugation/deconjugation equilibrium is affected by various environmental stresses, including osmotic, hypoxic, heat, oxidative and genotoxic stresses. This regulation occurs either at the level of individual targets, through an interplay between stress-induced phosphorylation and SUMOylation, or via modulation of the conjugation/deconjugation machinery abundance or activity. The present review gives an overview of the connections between stress and SUMOylation, the underlying molecular mechanisms and their effects on cellular functions.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling.

Cerebellar granule cell survival and maturation induced by K+ and NMDA correlate with c-fos proto-oncogene expression

Persistent depolarization with a high K+ concentration (30 mM) or sustained activation of N-methyl-D-aspartate (NMDA) receptors (12.5 mM K+ plus 100 microM NMDA) enhance both survival and maturation of mouse cerebellar granule neurons in vitro taking as criteria the amount of protein and DNA and the release of endogenous glutamate respectively. K+ and NMDA neurotrophic effects are associated with c-fos protein expression in the nucleus of these cells suggesting that c-fos protein could play a role in the survival and/or maturation of granule neurons.