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Physiological processes are driven by the coordinated action of organs, tissues and cells. Coordination of these activities allows organisms to respond appropriately to their changing environment. Within cells themselves, important metabolic processes are often compartmentalised by membranes, which necessitates the flow of information between compartments. How is this information integrated into the appropriate downstream response? We try to understand the basic principles that govern the flow of information in cells with particular emphasis on the role of lipid second messengers. By understanding the mechanisms by which these signals are transduced and propagated, we can better answer why and how things go wrong in disease.
We study how lipids regulate the activity of signaling enzymes, including both protein kinases and phosphatases. We use biochemical and cell biological assays supported by biophysical and structural techniques to deduce how, where, and when the signal is transduced. Our work is underpinned by quantitative biochemistry performed with precisely defined macromolecules. The insights we derive have the potential to rationalize disease pathogenesis at the molecular and atomic levels. For example, in the pro-growth and survival kinase Akt, we have characterized the mechanism by which a mutation associated with cancer and overgrowth disorders leads to kinase activity that is both spatially and temporally de-regulated. Determining how specific mutations drive the development of disease is essential for effective therapeutic intervention.
Thomas studied Biochemistry at the University of Bristol, U.K. before obtaining a PhD in Structural Biology at the Laboratory of Molecular Biology in Cambridge. Thomas moved to the USA in 2005 as an EMBO postdoctoral fellow at the National Institutes of Health in Bethesda, MD and started his own lab as an independent investigator at the Max Perutz Labs in 2012.
We recently demonstrated that the activity of a protein kinase called Akt, which promotes cell growth and proliferation, is strictly governed by the engagement of two specific lipids in cell membranes. Bypassing this requirement leads to uncontrolled growth and cancer.
Rho-associated coiled-coil kinase (ROCK) is a key signal transducer in regulating the cytoskeleton. Substrate engagement is controlled by the precise positioning of its kinase domains, which is achieved via a long coiled-coil domain that bridges its membrane binding domains to its kinase domains.
The protein kinase Akt integrates signals from membrane-embedded lipid second messengers and signals in the form of phosphorylation by upstream protein kinases. We have shown that one without the other is insufficient for Akt activation and that loss of the lipid signal triggers rapid dephosphorylation and inactivation of Akt. Akt is therefore switched on at the membrane and switched off in the cytosol.
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.
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.
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.
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).
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).
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.
Conformational sampling of membranes by Akt controls its activation and inactivation.
Lučić, Iva; Rathinaswamy, Manoj K; Truebestein, Linda; Hamelin, David J; Burke, John E; Leonard, Thomas A
A switch in nucleotide affinity governs activation of the Src and Tec family kinases
von Raußendorf, Freia; de Ruiter, Anita; Leonard, Thomas A.
PI(3,4,5)P3 Engagement Restricts Akt Activity toCellular Membranes
Michael Ebner, Iva Lučić, Thomas A. Leonard, and Ivan Yudushkin
Project title: “Structure, Function, and Regulation of Protein Kinase D" (P 30584)”
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)
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).
Project title: "Lipid-activated kinases in cell shape and motility" (P 28135)
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