Biochemistry & Biophysics | Molecular Cell Biology | Structural & Computational Biology
Structural Biology of Lipid-Activated Signal Transduction
Membranes are sites of intense signaling activity in eukaryotic cells. Essential processes such as autophagy, cytokinesis, exo- and endo- cytosis, and cytoskeletal remodeling depend on signal propagation at cellular...more
Membranes are sites of intense signaling activity in eukaryotic cells. Essential processes such as autophagy, cytokinesis, exo- and endo- cytosis, and cytoskeletal remodeling depend on signal propagation at cellular membranes. Dysregulation of signal transduction at these sites is the cause of a number of hereditary and non-hereditary diseases, including Coffin-Lowry syndrome, spinocerebellar ataxia, myotonic dystrophy, and various cancers. Over 500 kinases and 130 phosphatases regulate signal transduction by phosphorylating or dephosphorylating their target proteins. Given that the chemistry of phosphoryl transfer is conserved, there is a clear need for compartmentalization of what are essentially the same chemical reactions.
Signal Transduction by Lipid Second Messengers
One of the most important consequences of the activation of cell surface receptors is the generation of small molecule second messengers. In addition to the freely diffusible second messengers such as cAMP and inositol-1,4,5-triphosphate (IP3), a number of cellular second messengers are lipids. Despite being of fundamental importance to the exquisite spatial and temporal regulation of many cellular processes, the molecular mechanisms of lipid-mediated signal transduction are not well understood. Our goal is to understand how lipid second messengers can turn on signaling pathways at the membrane. To achieve this, we are using a spectrum of biophysical (including X-ray crystallography), biochemical, and cell biological techniques.
Akt – a Logic Gate Signal Processor
Many of the lipid responsive human protein kinases belong to the AGC family of kinases, of which paradigmatic lipid-activated kinases are Akt and protein kinase C (PKC). In 2017 we demonstrated the allosteric activation of Akt by the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2 for the first time. A mutation in the kinase domain associated with cancer and overgrowth disorders of the brain causes Akt hyperactivation by relieving autoinhibition (Ebner and Lučić et al., Molecular Cell 2017). In our most recent work on Akt, we describe the mechanism of activation in more detail and identify the allosteric coupling between PH domain-mediated autoinhibition and the inactivation of Akt by dephosphorylation (Lučić et al., PNAS 2018; Leonard [Reply to Letter to Editor], PNAS 2018). Conceptually, Akt can be thought of as an AND gate that integrates multiple signals to drive the biological response.
Switching Off with ADP
Fundamentally, we aim to describe signal transduction processes with deep mechanistic insight. By combining structure with quantitative in vitro biochemistry on rigorously validated samples, we can probe the structure-function relationship in absolute terms. We have recently applied these principles to study the Tec and Src family tyrosine kinases, work which has revealed a previously unknown switch in nucleotide binding that drives kinase activation (von Raußendorf et al., Sci Rep 2017).
Probing Structure In Vivo
Protein kinase C (PKC) is regulated by multiple membrane binding domains that respond to calcium, phospholipids, and the lipid second messenger diacylglycerol (DAG). PKC is held in an inactive conformation in resting cells by a complex network of intramolecular interactions that sequester its membrane binding domains and prevent substrate binding by its kinase domain. Using a dual color in vivo membrane translocation assay, we have shown that the ten mammalian PKCs adopt a conserved three-dimensional architecture despite domain rearrangements in their primary sequences (Lučić et al., Journal of Molecular Biology, 2016).
Membrane Scaffolding of Signaling Pathways
We are also interested in how signal transduction pathways are organized. Scaffolding of signaling proteins in the same pathway enhances specificity, promotes signal amplification by reducing noise, and, ultimately, improves signal propagation through the pathway. Membranes act as the scaffolds for many signaling reactions, including those involved in regulation of the actin cytoskeleton (Truebestein et al., Nature Communications 2016). Our studies are aimed at understanding how diverse signals are integrated, how substrate specificity is encoded not just at the kinase level, and the influence of the membrane environment on multi-component signaling hubs. This is an exciting area of research with frontiers in ageing, cancer, metabolic diseases such as diabetes, and obesity.
The Leonard lab invites applications from bright young people fascinated by science. If you are interested in joining a team to work on fundamental questions in biological signal transduction, please get in touch! For further details, please see: https://www.training.vbc.ac.at/phd-programme/
Congratulations to Kathi on a successful Master defence! Now for the PhD...
Congratulations to Iva on winning a 2018 University of Vienna Doc Award for her outstanding PhD thesis.
2017 VBC PhD Award to Iva for her outstanding PhD thesis. Well deserved!
Congratulations Dr. Lučić on a successful PhD defence!
Congratulations to Linda on being awarded a Hertha Firnberg Postdoctoral Fellowship!
Congratulations Dr. von Raußendorf on a successful defence!
Congratulations to Daniel on travel scholarship to EMBO meeting!
Congratulations Iva on being awarded a Univie Thesis Completion Fellowship!
Congratulations to Iva on her Keystone short talk invitation!
Congratulations to Iva on travel scholarship to EMBO meeting!
Leonard, Thomas A (2018). Reply to Agarwal: Activity against nuclear substrates is not necessarily mediated by nuclear Akt. P NATL ACAD SCI USA. PMID: 29907610
Lučić, Iva; Rathinaswamy, Manoj K; Truebestein, Linda; Hamelin, David J; Burke, John E; Leonard, Thomas A (2018). Conformational sampling of membranes by Akt controls its activation and inactivation. P NATL ACAD SCI USA. PMID: 29632185
von Raußendorf, Freia; de Ruiter, Anita; Leonard, Thomas A. (2017). A switch in nucleotide affinity governs activation of the Src and Tec family kinases Scientific reports.;7(1):17405. PMID: 29234112
National Scientific Research Fund (FWF) Project
Project title: "Structure, Function, and Regulation of Protein Kinase D" (P 30584)
Hertha Firnberg Postdoctoral Fellowship (FWF)
Dr. Linda Trübestein is a recipient of a Hertha Firnberg Postdoctoral Fellowship (FWF).
Project title: "Bridging the Gap - Regulation of the Cytoskeleton by the DMPK kinases" (T 915)
Doctoral Program "Signaling Mechanisms in Cellular Homeostasis"
The Leonard Group is a member of the special doctoral program "Signaling Mechanisms in Cellular Homeostasis", reviewed and funded by the Austrian Research Fund FWF (start 2017).
National Scientific Research Fund (FWF) Project
Project title: "Lipid-activated kinases in cell shape and motility" (P 28135)
Boehringer Ingelheim Fonds PhD Fellowship
Daniel Elsner is a recipient of the prestigious BIF PhD Fellowship.
Freia von Raussendorf is a recipient of the Austrian Academy of Sciences DOC Fellowship
Freia von Raußendorf, PhD defence 2017
Iva Lučić, PhD defence 2017