MST2-RASSF protein-protein interactions through SARAH domains.
ABSTRACT: The detailed, atomistic-level understanding of molecular signaling along the tumor-suppressive Hippo signaling pathway that controls tissue homeostasis by balancing cell proliferation and death through apoptosis is a promising avenue for the discovery of novel anticancer drug targets. The activation of kinases such as Mammalian STE20-Like Protein Kinases 1 and 2 (MST1 and MST2)-modulated through both homo- and heterodimerization (e.g. interactions with Ras association domain family, RASSF, enzymes)-is a key upstream event in this pathway and remains poorly understood. On the other hand, RASSFs (such as RASSF1A or RASSF5) act as important apoptosis activators and tumor suppressors, although their exact regulatory roles are also unclear. We present recent molecular studies of signaling along the Ras-RASSF-MST pathway, which controls growth and apoptosis in eukaryotic cells, including a variety of modern molecular modeling and simulation techniques. Using recently available structural information, we discuss the complex regulatory scenario according to which RASSFs perform dual signaling functions, either preventing or promoting MST2 activation, and thus control cell apoptosis. Here, we focus on recent studies highlighting the special role being played by the specific interactions between the helical Salvador/RASSF/Hippo (SARAH) domains of MST2 and RASSF1a or RASSF5 enzymes. These studies are crucial for integrating atomistic-level mechanistic information about the structures and conformational dynamics of interacting proteins, with information available on their system-level functions in cellular signaling.
Project description:RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs.
Project description:The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.
Project description:Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children's cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analysed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7-RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs.
Project description:Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 3??-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.
Project description:Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.
Project description:In eukaryotic cells, apoptosis and cell cycle arrest by the Ras --> RASSF --> MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long alpha-helices (h2/h2') provides an elongated binding interface between the two monomers, and the short 3(10) helices (h1/h1') are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.
Project description: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.
Project description:Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation . Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation . This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity . In this study, we show that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, we observe that dRASSF also possesses a tumor-suppressor function.
Project description:Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-? and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-?-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-?-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-?- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-?-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.
Project description:Ras proteins play a direct causal role in human cancer with activating mutations in Ras occurring in approximately 30% of tumours. Ras effectors also contribute to cancer, as mutations occur in Ras effectors, notably B-Raf and PI3-K, and drugs blocking elements of these pathways are in clinical development. In 2000, a new Ras effector was identified, RAS-association domain family 1 (RASSF1), and expression of the RASSF1A isoform of this gene is silenced in tumours by methylation of its promoter. Since methylation is reversible and demethylating agents are currently being used in clinical trials, detection of RASSF1A silencing by promoter hypermethylation has potential clinical uses in cancer diagnosis, prognosis and treatment. RASSF1A belongs to a new family of RAS effectors, of which there are currently 8 members (RASSF1-8). RASSF1-6 each contain a variable N-terminal segment followed by a Ras-association (RA) domain of the Ral-GDS/AF6 type, and a specialised coiled-coil structure known as a SARAH domain extending to the C-terminus. RASSF7-8 contain an N-terminal RA domain and a variable C-terminus. Members of the RASSF family are thought to function as tumour suppressors by regulating the cell cycle and apoptosis. This review will summarise our current knowledge of each member of the RASSF family and in particular what role they play in tumourigenesis, with a special focus on RASSF1A, whose promoter methylation is one of the most frequent alterations found in human tumours.