Leonard

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/

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