María A García

MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway

MT1‐matrix metalloproteinase (MT1‐MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1‐MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1‐MMP occurs during MDA‐MB‐231 adenocarcinoma cell migration into collagen type I three‐dimensional matrices. Polarized trafficking of MT1‐MMP is triggered by β1 integrin‐mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1‐MMP within VSV‐G/Rab8‐positive vesicles, but not in Rab11/Tf/TfRc‐positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1‐MMP exocytic traffic, collagen degradation and invasion, whereas Rab8‐ but not Rab11‐knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1‐MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1‐MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

TRAF Family Proteins Link PKR with NF-κB Activation

ABSTRACT The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase.

The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-κB activation

The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-κB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-κB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-κB induction. In PKR0/0 cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-κB. An inactive PKR mutant (K296R), was unable to induce NF-κB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-κB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2α phosphorylation and NF-κB activation. However, PKR mutants unable to activate NF-κB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-κB.

Resistance to viral infection of super p53 mice

Induction of expression of the tumor suppressor p53 after interferon treatment has been recently demonstrated (Takaoka et al., 2003), suggesting an antiviral activity of the protein. In addition, a direct correlation between p53 levels and tumor resistance has been addressed by generating mice with an extra copy of p53 (‘super p53’ mice) (Garcia-Cao et al., 2002). Here, we show that vesicular stomatitis virus replication in mouse embryo fibroblasts derived from ‘super p53’ mice is impaired as a result of apoptosis induction via p53 activation. These findings unequivocally demonstrate an antiviral activity of p53, a process that may contribute to inhibit the spread of the virus in vivo.

The impact of PKR activation: from neurodegeneration to cancer

An inverse association between cancer and neurodegeneration is plausible because these biological processes share several genes and signaling pathways. Whereas uncontrolled cell proliferation and decreased apoptotic cell death governs cancer, excessive apoptosis contributes to neurodegeneration. Protein kinase R (PKR), an interferon‐inducible double‐stranded RNA protein kinase, is involved in both diseases. PKR activation blocks global protein synthesis through eIF2α phosphorylation, leading to cell death in response to a variety of cellular stresses. However, PKR also has the dual role of activating the nuclear factor κ‐B pathway, promoting cell proliferation. Whereas PKR is recognized for its negative effects on neurodegenerative diseases, in part, inducing high level of apoptosis, the role of PKR activation in cancer remains controversial. In general, PKR is considered to have a tumor suppressor function, and some clinical data show a correlation between suppressed or inactivated PKR and a poor prognosis for several cancers. However, other studies show high PKR expression and activation levels in various cancers, suggesting that PKR might contribute to neoplastic progression. Understanding the cellular factors and signals involved in the regulation of PKR in these age‐related diseases is relevant and may have important clinical implications. The present review highlights the current knowledge on the role of PKR in neurodegeneration and cancer, with special emphasis on its regulation and clinical implications.—Marchal, J. A., Lopez, G. J., Peran, M., Comino, A., Delgado, J. R., García‐García, J. A., Conde, V., Aranda, F. M., Rivas, C., Esteban, M., Garcia, M. A. The impact of PKR activation: from neurodegeneration to cancer. FASEB J. 28, 1965–1974 (2014). www.fasebj.org

Anti-apoptotic and oncogenic properties of the dsRNA-binding protein of vaccinia virus, E3L

The vaccinia virus (VV) E3L gene encodes a dsRNA binding protein that inhibits activation of the IFN-induced, dsRNA-dependent protein kinase, (PKR), the 2–5A synthetases/RNase L system and other dsRNA dependent pathways, thus leading to efficient VV replication. To analyse E3L effects over cellular metabolism in a virus-free system, we have generated stable mouse 3T3 cell lines expressing E3L. Expression of E3L in NIH3T3 cells results in inhibition of eIF-2α phosphorylation and IκBα degradation in response to dsRNA. Antiviral responses induced by IFN-α/β were partially impaired in 3T3-E3L cells, as determined by a viability assay upon VSV infection. E3L expression also confers resistance to dsRNA-triggered apoptosis. Interestingly, cells expressing E3L grew faster than control cells, and showed increased expression of cyclin A and decreased levels of p27Kip1. E3L cooperated with H-ras in a focus formation assay, and NIH3T3 E3L cells formed solid tumors when injected in nude mice. Overall, our findings reveal that interference of E3L protein with several cellular pathways, results in promotion of cellular growth, impairment of antiviral activity and resistance to apoptosis.

Caspase 9 activation by the dsRNA-dependent protein kinase, PKR: molecular mechanism and relevance

The double‐stranded RNA‐dependent protein kinase (PKR) induces apoptosis by activation of the FADD/caspase 8 pathway. Here we show that upon PKR expression, caspase 9 is processed and activated, correlating with the translocation of cytochrome c to the cytoplasm and breakdown of mitochondrial potential upon Bax insertion. However, treatment of cells with an inhibitor of caspase 9 could not prevent PKR‐induced apoptosis. During PKR‐induced apoptosis, caspase 9 is activated downstream of caspase 8. Our findings revealed that caspase 9, although dispensable, is a mediator of PKR‐induced cell death.

SIRT1 Stabilizes PML promoting its Sumoylation

SIRT1, the closest mammalian homolog of yeast Sir2, is an NAD+-dependent deacetylase with relevant functions in cancer, aging, and metabolism among other processes. SIRT1 has a diffuse nuclear localization but is recruited to the PML nuclear bodies (PML-NBs) after PML upregulation. However, the functions of SIRT1 in the PML-NBs are unknown. In this study we show that primary mouse embryo fibroblasts lacking SIRT1 contain reduced PML protein levels that are increased after reintroduction of SIRT1. In addition, overexpression of SIRT1 in HEK-293 cells increases the amount of PML protein whereas knockdown of SIRT1 reduces the size and number of PML-NBs and the levels of PML protein in HeLa cells. SIRT1 stimulates PML sumoylation in vitro and in vivo in a deacetylase-independent manner. Importantly, the absence of SIRT1 reduces the apoptotic response of vesicular stomatitis virus-infected cells and favors the extent of this PML-sensitive virus replication. These results show a novel function of SIRT1 in the control of PML and PML-NBs.

Antiviral action of the tumor suppressor ARF

Oncogenic viruses frequently target the pathways controlled by tumor suppressor genes, suggesting an extra function for these proteins as antiviral factors. The control exerted by the tumor suppressor Arf on cellular proliferation is crucial to restrict tumor development; however, a potential contribution of Arf to prevent viral infectivity has remained unexplored. In the present study, we investigated the consequences of loss or increased expression of Arf on viral infection. Our results reveal that ARF expression is induced by interferon and after viral infection. Furthermore, we show that ARF protects against viral infection in a gene dosage‐dependent manner, and that this antiviral action is mediated in part by PKR through a mechanism that involves ARF‐induced release of PKR from nucleophosmin complexes. Finally, Arf‐null mice were hypersensitive to viral infection compared to wild‐type mice. Together, our results reveal a novel and unexpected role for the tumor suppressor ARF in viral infection surveillance.

Human Gene Profiling in Response to the Active Protein Kinase, Interferon-induced Serine/threonine Protein Kinase (PKR), in Infected Cells INVOLVEMENT OF THE TRANSCRIPTION FACTOR ATF-3 IN PKR-INDUCED APOPTOSIS

The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were down-regulated. The ATF-3 transcription factor, involved in stress-induced β-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VVΔE3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR-/- cells. Using null cells for ATF-3 and for the p65 subunit of NF-κB, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-κB activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis.