Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, we show that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, we demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress.

The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability.

The book edited by Gerry Melino and David Vaux functions as a compendium of cell death research, a journey that takes us back to the origins, while at the same time offers a glimpse into the future of research in this area. Cell demise is a crucial ‘event' in the regulation of development, tissue homeostasis and immune response. Dysregulation of cell death is known to contribute to human diseases, from cancer to autoimmune disorders. Although this book provides a useful source for experts in the field, it will also serve as a textbook for students at the undergraduate or postgraduate level who wish to explore the dynamics and regulation of cell death, from Caenorhabditis elegans to humans. As often the case with review books of this nature, there is a certain degree of repetition between chapters. This could have been avoided if the book was structured to follow a more defined line of thought. Nevertheless, each chapter contains novel information ensuring that this book stands out among the numerous others centred on cell death and constitutes an extremely useful addition.

Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function.

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.

The fourth p63/p73 workshop, supported by Epistem and Cell Death and Differentiation, was recently held in Toronto, Ontario, Canada at Mt. Sinai Hospital, organized by

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

c-Jun plays an important role in the cellular response to DNA damage.Following UV irradiation, c-Jun is rapidly induced and blocks p53-mediated upregulation of p21.c-Jun is required for re-entering the cell cycle following p53-mediated G1 arrest, 1 as c-Jun-null MEFs undergo a prolonged growth arrest following UV irradiation.Consequently, these cells show increased resistance to p53-dependent apoptosis. 1Moreover, c-Jun is also important for the DNA repair response following cisplatin treatment.Following DNA damage, c-Jun/ ATF complexes are rapidly recruited to the promoters of known DNA repair genes such as MSH2 and 6, Rad50 and ATM. 2 Although c-Jun promotes efficient DNA repair upon DNA damage, c-Jun also contributes to DNA damagemediated apoptosis.This is evident because c-Jun-deficient MEFs demonstrate enhanced resistance to cisplatin-induced apoptosis, 3,4 indicating a role of c-Jun in chemosensitivity.The cellular response to chemotherapy is critically dependent on functional p73.Reduction of p73 levels in tumour cells by siRNA, or the expression of dominant-negative mutants, leads to a strong reduction of apoptosis induced by DNA damaging agents such as cisplatin, camptothecin or doxorubicin. 5Importantly, this is irrespective of p53 status. 5 p73 function relies on the presence of Yes-Associated Protein (YAP).Like the loss of p73, downregulation of YAP similarly suppresses cisplatin-mediated cell death. 6,7ecent studies suggest that c-Jun promotes cell death by stabilising p73 following cisplatin treatment. 3Little is known regarding the precise molecular mechanism of c-Junmediated stabilisation of p73, although the fact that both the transactivation and DNA binding domains of c-Jun are required, suggests the existence of c-Jun targets with specific roles in cisplatin-induced apoptosis.Since YAP plays an important role in the p73-dependent response to cisplatin, we investigated whether YAP could be a possible mediator of c-Jun-dependent apoptosis.Sequence analysis of the YAP promoter revealed the presence of several putative AP-1 binding sites within 5000 bp relative to the transcriptional start site of YAP.Interestingly, using quantitative PCR (q-PCR) we found that YAP mRNA levels were induced in U2OS cells following transient transfection of c-Jun (Figure 1a).To validate these observations, we assessed YAP mRNA levels in c-Jun À/À MEFs and found that these cells expressed reduced YAP mRNA levels as compared to wild-type MEFs, and that retroviral-mediated with re-introduction of even small amounts of c-Jun restored YAP mRNA expression levels (Figure 1b).The levels of YAP mRNA correlated well with its protein expression.Thus, downregulation of c-Jun in U2OS cells using siRNA resulted in

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.