Nic Jones

YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides.

The role of the YAP1 transcription factor in the response of Saccharomyces cerevisiae cells to a variety of conditions that induce oxidative stress has been investigated. Cells deficient in YAP1 were found to be hypersensitive to hydroperoxides and thioloxidants, whereas overexpression of YAP1 conferred hyper‐resistance to the same conditions. These treatments resulted in an increase in YAP1‐specific binding to DNA together with an increase in YAP1 dependent transcription. Our results indicate that this increase is not due to an increase in synthesis of YAP1 protein, but rather results from modification of pre‐existing protein. Using a specific genetic screen, the TRX2 gene, one of two genes of S. cerevisiae that encode thioredoxin protein, was identified as being essential for YAP1 dependent resistance to hydroperoxides. Furthermore, efficient expression of TRX2 was dependent on YAP1 and enhanced under conditions of oxidative stress.

Regulation of yAP‐1 nuclear localization in response to oxidative stress

The YAP1 gene of Saccharomyces cerevisiae encodes a bZIP‐containing transcription factor that is essential for the normal response of cells to oxidative stress. Under stress conditions, the activity of yAP‐1 is increased, leading to the induced expression of a number of target genes encoding protective enzymes or molecules. We have examined the mechanism of this activation. Upon imposition of oxidative stress, a small increase in the DNA‐binding capacity of yAP‐1 occurs. However, the major change is at the level of nuclear localization; upon induction the yAP‐1 protein relocalizes from the cytoplasm to the nucleus. This regulated localization is mediated by a cysteine‐rich domain (CRD) at the C‐terminus, its removal resulting in constitutive nuclear localization and high level activity. Furthermore, the CRD of yAP‐1 is sufficient to impose regulated nuclear localization of the GAL4 DNA‐binding domain. Amino acid substitutions indicated that three conserved cysteine residues in the CRD are essential for the regulation. We suggest therefore, that these cysteine residues are important in sensing the redox state of the cell and hence regulating yAP‐1 activity.

Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins.

The passage of mammalian cells through the restriction point into the S phase of the cell cycle is regulated by the activities of Cdk4 and Cdk6 complexed with the D-type cyclins and by cyclin E/Cdk2 (refs 1,2,3). The activities of these holoenzymes are constrained by CDK inhibitory proteins. The importance of the restriction point is illustrated by its deregulation in many tumour cells and upon infection with DNA tumour viruses. Here we describe the properties of cyclins encoded by two herpesviruses, herpesvirus saimiri (HVS) which can transform blood lymphocytes and induce malignancies of lymphoid origin in New World primates, and human herpesvirus 8 (HHV8) implicated as a causative agent of Kaposis sarcoma and body cavity lymphomas. Both viral cyclins form active kinase complexes with Cdk6 that are resistant to inhibition by the CDK inhibitors p16Ink4a, p21Cip1and p27Kip1. Furthermore, ectopic expression of a viral cyclin prevents G1 arrest imposed by each inhibitor and stimulates cell-cycle progression in quiescent fibroblasts. These results suggest a new mechanism for deregulation of the cell cycle and indicate that the viral cyclins may contribute to the oncogenic nature of these viruses.

Redox control of AP-1-like factors in yeast and beyond

Cells have evolved complex and efficient strategies for dealing with variable and often-harsh environments. A key aspect of these stress responses is the transcriptional activation of genes encoding defense and repair proteins. In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress. This sub-family of AP-1 (called yAP-1) proteins are sensors of the redox-state of the cell and are activated directly by oxidative stress conditions. yAP-1 proteins are bZIP-containing factors that share homology to the mammalian AP-1 factor complex and bind to very similar DNA sequence sites. The generation of reactive oxygen species and the resulting potential for oxidative stress is common to all aerobically growing organisms. Furthermore, many of the features of this response appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility. The important structural features of these factors, signaling pathways controlling their activity and the nature of the target genes they control will be discussed.

Modulation of p27Kip1 levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposis sarcoma‐associated herpesvirus) which is able to resist the actions of p16Ink4a, p21Cip1 and p27Kip1 cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27Kip1. We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin–cdk2 complexes but not cyclin D1–cdk6. Included amongst these substrates is p27Kip1 which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27Kip1 downregulation, this being consistent with previous studies indicating that phosphorylation of p27Kip1 on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27Kip1 in which Thr187 is mutated to non‐phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27Kip1‐imposed G1 arrest by facilitating phosphorylation and downregulation of p27Kip1 to enable activation of endogenous cyclin–cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus‐encoded cyclin.

Stress‐activated signalling pathways in yeast

Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions. In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress‐protective functions. Over the last few years, considerable progress has been made in understanding how stress‐dependent transcriptional changes are brought about, and it transpires that the underlying mechanisms are highly conserved, being similar in organisms ranging from yeast to man. Many of the stress signals derive from the extracellular environment and accordingly these signals require transduction from the cell surface to the nucleus. This is accomplished through stress‐activated signalling pathways, key amongst which are the highly conserved stress‐activated MAP kinase pathways. Stimulation of these pathways leads to the increased activity of specific transcription factors and consequently the increased expression of certain stress‐related genes. In this review, we focus on the progress that has been made in understanding these stress responses in yeast.

Phosphorylation of E2F-1 modulates its interaction with the retinoblastoma gene product and the adenoviral E4 19 kDa protein

The transcription factor E2F is regulated through its cyclical interaction with a spectrum of cellular proteins. One such protein is the product of the retinoblastoma gene (Rb); association of E2F with Rb inhibits its transactivation potential. However, in adenovirus-infected cells, E2F is complexed to the 19 kDa product of the adenovirus E4 gene. We have studied the interaction of E2F-1 with the Rb and adenovirus E4 proteins and show that phosphorylation of E2F-1 on serine residues 332 and 337 prevented its interaction with Rb but was a prerequisite for interaction with E4. These residues were phosphorylated in vivo and by p34cdc2 kinase in vitro. Upon stimulation of serum-starved cells, phosphorylation was induced in the late G1 phase of the cell cycle. These observations suggest that phosphorylation of E2F-1 is important in the regulation of its activity during the cell cycle and during infection of cells by adenovirus.

AP-1 transcription factors in yeast

In the past two years, the completion of the Saccharomyces cerevisiae genome project and molecular analysis of other fungal species has resulted in the identification of a growing number of yeast AP-1 transcription factors. Characterisation of these factors indicates that, like their mammalian counterparts, they activate gene expression in response to a variety of extracellular stimuli. In particular, these factors are required for the response to oxidative stress and for surviving exposure to a variety of cytotoxic agents. Much progress has also been made in understanding how members of this family of proteins are regulated. These studies promise to further our awareness of eukaryotic stress responses and are likely to have implications for the study of mammalian AP-1.

Distinct Roles for Glutathione S-Transferases in the Oxidative Stress Response in Schizosaccharomyces pombe

We have identified three genes,gst1 + , gst2 + , andgst3 + , encoding θ-class glutathioneS-transferases (GSTs) in Schizosaccharomyces pombe. The gst1 + andgst2 + genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. Thegst1Δ, gst2Δ, and gst3Δ mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Bothgst2 + and gst3 + mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1 + ,gst2 + , and gst3 + are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast.

E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest

BACKGROUND The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle. This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130. RESULTS In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16Ink4, which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21Cip1 or p27Kip1 cannot be bypassed either by inactivation of pRb or overexpression of E2F family members. CONCLUSIONS These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F-pRb complex. The cyclin E-cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.