Stress Signalling Pathways

Stress Signalling Pathways describe the molecular activities emanating from (generally adverse) external stimuli that are received by cells via surface or internal receptors and transmitted via relays of protein kinases. Depending on the type of stimulus and cellular context, activation of these pathways may lead to induction of diverse cellular programmes, ranging from growth to growth arrest, differentiation, or cell death. 

 One such signalling pathway is described by the stress activated protein kinase (SAPK) pathway. Formally this has been categorised as a specific branch of the wider acting MAPK (mitogen activated protein kinase) pathways and involves a serious of protein kinases, which act as signalling molecules that transmit information via phosphorylation of their protein substrates. Thus, SAPKs (including members of the JNK and p38 subfamilies) are activated by upstream acting layers of kinases comprising SAPKKs (e.g. MAP2K3, 4, 6, and 7) and SAPKKK (e.g. MAP3K1, 2, etc.). At the base of this hierarchy lie cellular effector proteins that include other kinases, mitochondrial proteins, and transcription factors. Together, the actions of the effector molecules, in response to activation by the SAPKs determine the cellular response to a specific stimulus.

Stress Kinase pathways in cancer
Recent advances in cancer genome analyses have highlighted the frequency at which stress signalling pathways are deregulated, or mutated, in various tumour types. E.g. the signalling kinase MEKK1 (MAP3K1) is mutated in 9% of breast invasive and endometrial carcinoma and 6% in prostate adenocarcinoma (source: cbioportal.org), while its substrate, MAP2K4, is mutated in 6-7% of breast and colon cancers.  The nature of these mutations suggests that the pathway is aberrantly activated in some types of cancer, but to a far greater extent, inactivated in many other types. Thus, stress signalling pathways are thought to be pro-oncogenic or tumour suppressive depending on the cancer context.

ATF2: an effector transcription factor of stress kinase signalling
One focus of research in the Cell Regulation group is the AP-1 transcription factor ATF2, which is an effector substrate of the SAPKs, JNK and p38. Our research work has demonstrated that ATF2 has essential functions during development of the embryonic liver, heart and brain, and that this is dependent on its activation by the SAPK signalling cascade.

High levels of phospho-ATF2 have been detected in both human melanoma and prostate carcinoma samples, and a role for ATF2 in driving progression of these tumours has been suggested in the literature. For example, recent findings indicate that the SS18-SSX2 fusion protein, found in human synovial sarcomas, derives its oncogenicity from the ability to interact with ATF2. Conversely, low levels of ATF2 expression in human breast tumours have also been reported.

Functions of JNK and ATF2 in B lymphoma
Experimental analyses using mouse tumour models carried out by our group as well as by others, have uncovered diverse roles for ATF2 in tumourigenesis. Accordingly, ATF2 was shown to contribute to melanoma development through its role in a melanocyte-specific gene activation programme. In contrast, ATF2 deficient mice are sensitised to carcinogen-induced skin tumourigenesis underlining the tumour context dependent activities of the stress signalling pathway. In a mouse model of MYC oncogene induced B-cell lymphoma development, we showed that ATF2 deficiency results in accelerated disease onset. Further in vitro analysis revealed that ATF2 responds to oncogene induced cellular stress by inducing programmed cell death. In addition, JNK and ATF2 were shown to direct the induction of apoptosis in response to chemotherapeutic drugs but only once B lymphocytes have progressed towards lymphoma stages by the actions of cMYC. Thus, ATF2 is an effector of cMYC induced apoptosis through activation by JNK.

 

Figure 1. Gene Level Analysis: A heat map depicting differentially expressed genes in hepatoblasts in response to ATF2 activity. Expression array experiments were carried out in triplicate (columns) of control or ATF2 expressing cells. Over 250 up-regulated (red bars) and over 30 down-regulated (blue bars) genes were identified.

Tumour suppressive transcriptional programmes by ATF2
An independent project has focused on the role of SAPK pathway components in hepatocellular carcinoma. Here, we demonstrated that JNK dependent activation of ATF2 is critical in blocking the oncogenic transformation of hepatocyte precursors (hepatoblasts) as well as in suppressing their tumourigenicity after orthotopic transplantation into recipient livers.  In addition, we defined a JNK and ATF2 dependent transcriptional programme that acts in a tumour suppressive manner. Further analysis revealed that this programme is frequently found inactivated or genetically altered in a variety of human tumours types, including breast, lung, pancreatic and hepatocellular carcinoma. This analysis therefore confirmed that the experimental tumour models reflect human cancer scenarios. Further analysis of SAPK dependent effectors as well as other components of stress signalling pathways will therefore be the focus of future research.

Identification of novel targets of the S. pombe MAP kinase Sty1
The SAPK pathways are highly conserved between yeast and mammalian cells, and in Schizosaccharomyces pombe the MAPK Sty1 is a key regulator of the stress response. Sty1 is activated following the exposure of cells to a wide variety of environmental stress conditions. Similarly to the mammalian SAPKs, Sty1 is activated through dual phosphorylation by an upstream MAPKK. Due to the high level of conservation between the mammalian and yeast signalling pathways, S. pombe provides an ideal model to investigate the function and regulation of the SAPK signalling pathways.

Upon activation Sty1 mediates the appropriate stress response through the phosphorylation of downstream target proteins, the best characterised of which is the transcription factor Atf1. Whilst only a small number of Sty1 targets are currently known, the large number of cellular processes regulated by the Sty1 pathway suggests that there are likely to be a number of, as yet, uncharacterised Sty1 target proteins. To fully understand the stress response, it is vital that we identify targets of SAPKs.

 

Figure 2. The S. pombe Sty1 MAPK signalling pathway. The Sty1 kinase is activated by a kinase cascade. The activated MAPK Sty1 then phosphorylates a range of target proteins to mediate the stress response.

In order to identify novel targets of Sty1 phosphorylation we performed a SILAC screen in collaboration with the laboratory of Boris Maček at the University of Tübingen. Intriguingly, we found that following exposure to oxidative stress, proteins upstream of Sty1 in the MAPK pathway are phosphorylated in a Sty1-dependent manner. Furthermore, one such protein is a potential direct target of Sty1 phosphorylation. Further investigation revealed that Sty1-dependent phosphorylation of upstream components appears to promote Sty1 phosphorylation, thus forming a positive feedback loop to increase the level of Sty1 activity following oxidative stress exposure.