Molecular Mechanisms of 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 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 structural biology (including cryo-electron microscopy and X-ray crystallography), biochemical, and cell biological techniques. 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.

Many of the lipid responsive human protein kinases belong to the AGC family of kinases, of which paradigmatic lipid-activated kinases are Akt, PDK1 and Sgk3. 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). We have since described the mechanism of activation in more detail (Truebestein et al., PNAS 2021) and characterized the allosteric coupling between PH domain-mediated autoinhibition and the inactivation of Akt by dephosphorylation (Lučić et al., PNAS 2018). Conceptually, Akt can be thought of as an AND gate that integrates multiple signals to drive the biological response.

Similarly, Sgk3 employs similar mechanisms of autoinhibition and allosteric activation to drive signaling downstream of the lipid second messenger PI3P. Like Akt, Sgk3 exhibits an inactive conformation in the absence of PI3P in which the PI3P-binding pocket is sequestered in an intramolecular interface between the regulatory and kinase domains (Pokorny et al, Journal of Biological Chemistry 2021). Akt and Sgk3 activities are thereby restricted, exclusively, to those membranes in the cell where they can be activated by their cognate lipids. 

Whilst much of our work has concerned protein kinases, widely understood to be the ‘on’ switches in cell signaling, the phosphatases that reverse the phosphorylation of proteins are equally as important. Our work on Akt activation immediately begged an answer to the question of how Akt is inactivated. We therefore began work on a protein called PH domain leucine-rich repeat-containing protein phosphatase (PHLPP), which has been proposed to inactivate Akt and, thereby, act as a tumor suppressor in cells. In an ironic twist, given that this was our first venture into the phosphatase field, we discovered that PHLPP is neither a phosphatase nor a tumor suppressor (Husremović & Meier et al, PNAS 2025). In fact, while PHLPP is descended from an ancestral phosphatase formed more than 1.5 Mya, it lost activity in the last metazoan ancestor. Structural phylogenomics shows that the ancestral gene was duplicated, neo- and sub-functionalized, and reciprocal gene loss in fungi and metazoans led to the repurposing of the protein in different signaling pathways. We are now focused on elucidating the biological functions of mammalian PHLPP, the ancestral phosphatase, and the fungal homolog, CYR1.

Protein kinases are often activated by autophosphorylation of a key residue in their activation loop that drives catalytic activity. Mechanistically, this reaction may occur in trans or in cis. In 2019, we determined the structure of a novel ubiquitin-like domain in Protein Kinase D (PKD) which mediates its dimerization (Elsner et al., Journal of Biological Chemistry 2019). PKD, however, is maintained in an inactive conformation by dimerization and relief of this trans-autoinhibition leads to activation loop autophosphorylation in cis  (Reinhardt et al., PNAS 2023). PKD is an essential mammalian kinase involved in trafficking of cargo destined for secretion from the trans-Golgi network to the plasma membrane. 

In a parallel study on phosphoinositide-dependent kinase 1 (PDK1), which is a master regulator of growth factor signaling, we have determined the mechanism of its activation by PIP₃-elicited dimerization and trans-autophosphorylation (Levina et al., Nature Communications 2022). These studies showcase how nature has employed common strategies, such as dimerization and activation loop phosphorylation, in different ways to control signal transduction.

In work on the Tec and Src family tyrosine kinases, we have revealed a previously unknown switch in nucleotide binding that drives kinase activation  (von Raußendorf et al., Sci Rep 2017). Here nature utilizes ADP, the product of the kinase reaction, to stabilize and maintain the inactive conformation. Upon triggering of the conformational changes required for kinase activation, these kinases lose their strict preference for ADP, which is then rapidly exchanged for ATP in the cell. The binding of ADP in the inactive conformation serves to insulate the kinase from promiscuous activity, while activation drives the exchange of ADP for the ATP required for substrate phosphorylation.

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; Truebestein et al., Structure 2023). 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.