Comparative Analysis of Interactions of the RASSF Family Mediated by the Ras-association (RA) and SARAH domains

Members of the RASSF family (RASSF1-10) have been identified as candidate tumour suppressors that are frequently downregulated by promoter hypermethylation in cancers. These adaptor proteins carry a common Ras-association (RA) and SARAH domain (RASSF1-6) that can potentially bind Ras oncoproteins and mediate protein-protein interactions with other SARAH domain proteins (e.g. MST kinase). However, there is a notable lack of comparative characterisation of the RASSF family, as well as of molecular and structural information that facilitate their tumour suppressive functions. As part of our comparative analysis, we modelled the RA and SARAH domains of the RASSF members based on existing structures and predicted their potential interactions and the key residues involved. These in silico predictions were compared to in vitro studies and intracellular binding assays using Förster Resonance Energy Transfer (FRET). Several SARAH domain mutants were also investigated for their effects on RASSF interactions. Furthermore, we compared the interactions of the RASSF family with several key proteins involved in death and NFκB signalling. Our biochemical data show a diversity of interactions within the RASSF family RA domain, whereas interactions between RASSF and MST correlate with the presence of the SARAH domain, which is supported by the FRET experiments. Mutations of specific non-polar residues in the dimerisation interface of the SARAH domain also prove detrimental to the interaction between selected RASSF members and MST. Moreover, we observed stimulation-dependent interactions between specific RASSF members and MOAP1, TNF-R1, DAPK and TBK1. These results suggest that different members, despite shared general architecture, may have distinct binding properties, but ultimately could share overlapping functions. Current data also support an interaction model where RASSF serves as an adaptor for the assembly of multiple protein complexes and further functional interactions, involving MST kinases and other interacting partners, which could be regulated by Ras

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system

RASSF Signalling and DNA Damage: Monitoring the Integrity of the Genome?

The RASSF family of proteins has been extensively studied in terms of their genetics, structure and function. One of the functions that has been increasingly studied is the role of the RASSF proteins in the DNA damage response. Surprisingly, this research, which encompasses both the classical and N-terminal RASSF proteins, has revealed an involvement of the RASSFs in oncogenic pathways as well as the more familiar tumour suppressor pathways usually associated with the RASSF family members. The most studied protein with respect to DNA damage is RASSF1A, which has been shown, not only to be activated by ATM, a major regulator of the DNA damage response, but also to bind to and activate a number of different pathways which all lead to and feedback from the guardian of the genome, p53. In this review we discuss the latest research linking the RASSF proteins to DNA damage signalling and maintenance of genomic integrity and look at how this knowledge is being utilised in the clinic to enhance the effectiveness of traditional cancer therapies such as radiotherapy

RASSF1A Signaling in the Heart: Novel Functions beyond Tumor Suppression

The RASSF proteins are a family of polypeptides, each containing a conserved Ras association domain, suggesting that these scaffold proteins may be effectors of activated Ras or Ras-related small GTPases. RASSF proteins are characterized by their ability to inhibit cell growth and proliferation while promoting cell death. RASSF1 isoform A is an established tumor suppressor and is frequently silenced in a variety of tumors and human cancer cell lines. However, our understanding of its function in terminally differentiated cell types, such as cardiac myocytes, is relatively nascent. Herein, we review the role of RASSF1A in cardiac physiology and disease and highlight signaling pathways that mediate its function

Mst1/2 signalling to Yap: gatekeeper for liver size and tumour development

The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans

Using BioID to study RAS signaling to the Hippo pathway

RAS est une GTPase qui transduit les signaux extracellulaires envers des voies de signalisation intracellulaires, en liant ses effecteurs. RAS peut activer la voie Hippo qui inhibe la croissance cellulaire et qui est souvent dérégulée dans le cancer. Les protéines RASSF suppresseurs de tumeurs relient RAS à la voie Hippo. L’expression exogène de KRASG12V avec RASSF1 ou RASSF5 conduit à l'activation de la voie Hippo, bien que KRAS et RASSF1 ne s’associent pas directement. Ce projet de maîtrise vise à identifier les protéines impliquées dans l'activation de la voie Hippo par RAS. Nous avons effectué plusieurs expériences BioID, une technique qui permet d’identifier les interacteurs proximaux d’une protéine d’intérêt, dans des lignées cellulaires U2OS stables et inductibles exprimant les protéines KRASG12V, RASSF1 ou RASSF5 seules ou coexprimées, permettant de comparer les conditions où la voie Hippo inactive ou active. Nous avons élucidé l'interactome d'un mutant de KRAS avec affinité accrue envers RASSF5 et affinité réduite envers RAF, permettant d’étudier les voies activées en aval de RASSF5, avec une activation réduite de la voie MAPK. Nos données montrent que RASSF1 et RASSF5 relâchent les kinases Hippo MST1 et MST2 lorsque la voie Hippo est active, conformément aux données in vitro démontrant un rôle inhibiteur de l'interaction RASSF/MST. De plus, nous avons démontré que KRAS est un interacteur proximal des protéines VAMP3 et SNAP23. Comprendre comment l'oncoprotéine RAS active des effecteurs et des voies de signalisation moins étudiés, en particulier ceux qui ont des fonctions suppressives de tumeurs a des implications importantes pour le développement de nouvelles thérapies ciblées pour les cancers induits par RAS.RAS is a small GTPase that transduces signals from membrane-bound receptors to intracellular pathways, by signaling to downstream effector proteins. RAS can activate the Hippo pathway, a growth-suppressive pathway that is often dysregulated in cancer. The tumor suppressor RASSF proteins link RAS to Hippo signaling. Co-expression of KRASG12V with either RASSF1 or RASSF5 leads to Hippo pathway activation, despite KRAS and RASSF1 not being direct binding partners. This M.Sc. project aims to identify proteins that are involved in RAS-mediated activation of the Hippo pathway. We performed BioID, a proteomic technique which is used to identify the proximal interactors of a protein of interest, in stable and inducible U2OS osteosarcoma lines expressing KRASG12V, RASSF1, or RASSF5 proteins alone, as well as in lines co-expressing both KRASG12V and the RASSF proteins, allowing for a comparison between inactive and active Hippo pathway interactomes. Furthermore, we mapped the interactome of a double mutant of KRAS that displays increased affinity for RASSF5 and decreased affinity for the effector RAF, allowing us to study KRAS signaling downstream of RASSF5, with decreased activation of the MAPK pathway. Our BioID data shows that RASSF1 and RASSF5 disengage the Hippo kinases MST1 and MST2 when the Hippo pathway is active, in line with the inhibitory role of the RASSF/MST interaction observed in vitro. Furthermore, we show that KRAS is a proximal interactor of the SNARE proteins VAMP3 and SNAP23. Understanding how the oncoprotein RAS signals to less studied effectors and pathways, particularly those with tumor suppressive functions has significant implications for understanding oncogenesis, and for development of new targeted therapies for RAS-driven cancers

Optimization of Techniques for Visualizing Protein Functionality in Cell Culture

The Hippo pathway is a highly conserved signaling pathway responsible for the regulation of cellular growth, proliferation and apoptosis. The Drosophila pathway is made up of four key proteins Salvador, Hippo, Warts and Mats (Salvador, MST1/2, Lats1/2 and Mob in mammals, respectively). At the heart of the signaling cascade are the two core kinases Hippo(MST) and Warts(Lats). In order for the pathway to function both of these kinases must be phosphorylated and active. Ultimately, Hippo(MST) will phosphorylate Warts(Lats). Once phosphorylated, Warts(Lats) is active and proceeds to phosphorylate its target Yorkie(YAP). Phosphorylation of Yorkie(Yap) sequesters the transcriptional co-activator from the nucleus leading to a downregulation of pro-growth genes. The function of the Hippo pathway core proteins in cultured cells can be monitored using luciferase-based reporter assay where luciferase signal is a proxy for Yorkie/YAP localization. I have adapted existing protocols to use this assay in both human (HEK293T) and insect (S2) cell lines in the Kavran Lab. Once established, I used the luciferase assay to help elucidate the role of the linker region and phosphorylation sites of MST2 in kinase activity, define the minimal functional unit of Lats2, validate the biological significance of cysteine 624 in Hippo, and learn about the role of SARAH domains in protein binding and activity. Additionally, preliminary work has been started to investigate the “hierarchy” of SARAH domains and to validate the SARAH domain interactions of dSalvador required for homodimer formation