Josée N. Lavoie

Modulation of Cellular Thermoresistance and Actin Filament Stability Accompanies Phosphorylation-Induced Changes in the Oligomeric Structure of Heat Shock Protein 27

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

Inhibition of Daxx-mediated apoptosis by heat shock protein 27.

ABSTRACT Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.

Expression of drosophila's 27 kDa heat shock protein into rodent cells confers thermal resistance

The role of hsp27, one of Drosophila melanogasters small heat shock proteins, in the process of thermotolerance was investigated. The coding sequence of hsp27 was subcloned downstream of the human hsp27 promoter which has been shown to be constitutively expressed in Chinese hamster O23 cells. Cellular resistance to a thermal stress was measured two days after transfection by a survival assay following a 3.5 h heat treatment at 44 degrees C. Expression of Drosophila hsp27 was shown to confer thermal resistance to O23 cells in a manner which was dependent on the level of expression of this hsp. Immunoblot analysis confirmed that the thermal resistance was related to the expression of Drosophila hsp27 as none of the endogeneous hsps showed an increased level under these conditions.

Genetic cooperation between the Werner syndrome protein and poly(ADP-ribose) polymerase-1 in preventing chromatid breaks, complex chromosomal rearrangements, and cancer in mice

Werner syndrome is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for Werner syndrome encodes a DNA helicase/exonuclease protein. Participation in a replication complex is among the several functions postulated for the WRN protein. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme, which is known to bind to DNA strand breaks, is also associated with the DNA replication complex. To determine whether Wrn and PARP-1 enzymes act in concert during cell growth, mice with a mutation in the helicase domain of the Wrn gene (Wrn(Deltahel/Deltahel) mice) were crossed to PARP-1-null mice. Both Wrn(Deltahel/Deltahel) and PARP-1-null/Wrn(Deltahel/Deltahel) cohorts developed more neoplasms than wild-type animals. The tumor spectrum was the same between PARP-1-null/Wrn(Deltahel/Deltahel) mice and Wrn mutants. However, PARP-1-null/Wrn(Deltahel/Deltahel) mice developed neoplasms at a younger age. Mouse embryonic fibroblasts derived from such PARP-1-null/Wrn(Deltahel/Deltahel) mice stop dividing abruptly unlike Wrn(Deltahel/Deltahel) or PARP-1-null cells. PARP-1-null/Wrn(Deltahel/Deltahel) fibroblasts were distinguished by an increased frequency of chromatid breaks, complex chromosomal rearrangements, and fragmentation. Finally, experiments have indicated that the PARP-1 enzyme co-immunoprecipitates with the WRN protein in human 293 embryonic kidney cells. These results suggest that Wrn and PARP-1 enzymes may be part of a complex involved in the processing of DNA breaks.

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane–cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.

The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.

The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G2/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.

Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

ABSTRACT In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Src-mediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.

Establishment and Characterization of a New Cell Line Derived from a Human Primary Breast Carcinoma

A new cell line, designated HDQ-P1, was successfully established from a primary ductal infiltrating mammary carcinoma by using a 3T3 feeder layer lethally irradiated to 60 Gy. The HDQ-P1 cells have been grown in culture for over 115 passages and have a doubling time of 60 hours. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, telomerase expression, tumor antigen expression, xenograft implantation into nude mice, colony formation in soft agar, TP53 sequencing, and gene copy number of C-MYC, C-ERBB-2, and C-H-RAS oncogenes. The epithelial nature of this cell line was confirmed by ultrastructural analysis, expression of cytokeratins, and epithelial membrane antigen. The HDQ-P1 cells possess an extensively rearranged and polyploid karyotype, with an average of 20 recurrent marker chromosomes. Scatchard analysis demonstrated that both primary tumor and HDQ-P1 cells were estrogen- and progesterone-receptor negative. The HDQ-P1 cells had the same expression of human telomerase reverse transcriptase as other established breast cancer cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Direct DNA sequencing showed a point mutation which yielded to a stop codon at the amino acid 213 in exon 6 of the TP53 gene. A five-fold amplification of C-MYC was observed in HDQ-P1 cells. No amplification of C-ERBB-2 and C-H-RAS genes were observed. This cell line presents unique characteristics and may prove to be a good experimental model for investigating breast cancer biology.

Nuclear localization of the adenovirus E4orf4 protein is mediated through an arginine-rich motif and correlates with cell death.

The adenovirus E4orf4 protein induces p53-independent death of human cancer cells by a mechanism requiring interactions with the Bα subunit of protein phosphatase 2A. When expressed alone E4orf4 localizes predominantly in the nucleus, although significant levels are also present in the cytoplasm. While tyrosine phosphorylation of E4orf4 and recruitment of Src have been linked with E4orf4 cytoplasmic cell death functions, little is known about the functions of E4orf4 in the nucleus. In this study, we identified an arginine-rich motif (E4ARM; residues 66–75) that is necessary and sufficient for nuclear and nucleolar localization. This motif, which is highly homologous to the arginine-rich nuclear and nucleolar localization motif of some lentiviral proteins, was shown to target heterologous proteins to the nucleus and to nucleoli, functions found to be dependent on the overall charge of the motif rather than on specific residues. Furthermore, mutation of arginine residues to alanines but not to lysines in E4ARM was shown to block such targeting activity and, when introduced into full-length E4orf4, to decrease induction of cell death. Finally, coexpression of the ARM motifs of E4orf4, HIV-1 Tat or Rev along with full-length E4orf4 was seen to decrease E4orf4-dependent cell killing. Thus it appears that targeting of E4orf4 to the nucleus and cell nucleoli by E4ARM is an important component of E4orf4-induced cell death.

Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases, Cdc42 and Rab11a

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.