Of the approximately 10000 proteins in a typical mammalian cell, about a third are phosphorylated at any given time. This reversible chemical modification controls the activity and localization of proteins and is therefore a key regulatory step in many cellular processes, from cell signaling to metabolism. The Leonard lab focuses on the molecular mechanisms of signal transduction. As part of their work, the scientists study protein kinases, enzymes that catalyze phosphorylation and, thereby, transduce and propagate signals within cells. In 2019, the Leonard lab discovered a ubiquitin-like domain (ULD) in PKD that mediates its dimerization. Since PKD was known to require phosphorylation in order to be activated, the scientists assumed that PKD must phosphorylate its own activation loop in trans. This dimerization-mediated trans model of kinase autoactivation has been observed in many auto-phosphorylating protein kinases, and thus seemed a logical conclusion.
But when PhD student Ronja Reinhardt started working on the project, she soon faced major challenges: "We had a clear hypothesis, but for a long time our assays didn’t work out the way we thought they would and the data just didn’t add up." The scientists soon figured out why. Counterintuitively, autophosphorylation of PKD was inhibited by dimerization. "Through a lot of critical thinking and careful experimentation, we came to understand that PKD is actually activated in completely the inverse mechanism to what we expected." In the scientists’ revised model, PKD is by default dimeric and locked in a compact, inactive conformation. When it binds to the membrane signaling lipid diacylglycerol, it undergoes conformational changes that force the kinase domains to dissociate from one another, thereby freeing them to phosphorylate themselves in cis. This means that each kinase domain phosphorylates its own activation loop.
The mechanistic details of the process that releases the protein from its locked conformation remain to be elucidated. The study also raises questions about the activation mechanisms of other protein kinases. "For me the most exciting aspect of the work is that it reveals a completely unexpected but extremely logical mechanism that nature has found to solve the same problem with a different solution", says group leader Thomas Leonard. The work also teaches a lesson about the scientific process itself, which the researchers are keen to point out: "Our work illustrates the importance of rigorously testing scientific hypotheses. A negative result is just as valuable as a positive one, and one should always rethink the hypothesis based on the experimental data, and not the other way around," Thomas Leonard concludes.
Ronja Reinhardt, Kai Hirzel, Gisela Link, Stephan A Eisler, Tanja Hägele, Matthew A H Parson, John E Burke, Angelika Hausser, Thomas A Leonard: PKD autoinhibition in trans regulates activation loop autophosphorylation in cis. PNAS 2023
Nutrient-regulated control of lysosome function by signaling lipid conversion
Shedding Light on the Dark Side of Terrestrial Ecosystems: Assessing Biogeochemical Processes in Soils
Protein homeostasis and lifelong cell maintenance
Dissecting the turgor sensing mechanisms in the blast fungus Magnaporthe oryzae
Pikobodies: What does it take to bioengineer NLR immune receptor-nanobody fusions
When all is lost? Measuring historical signals
Gene regulatory mechanisms governing human development, evolution and variation
Regulation of Cerebral Cortex Morphogenesis by Migrating Cells
Phage therapy for treating bacterial infections: a double-edged sword
Suckers and segments of the octopus arm
Using the house mouse radiation to study the rapid evolution of genes and genetic processes
CRISPR jumps ahead: mechanistic insights into CRISPR-associated transposons
SLiMs and SHelMs: Decoding how short linear and helical motifs direct PPP specificity to direct signaling
Title to be announced
Visualising mitotic chromosomes and nuclear dynamics by correlative light and electron microscopy
Enigmatic evolutionary origin and multipotency of the neural crest cells - major drivers of vertebrate evolution
Engineered nanocarriers for imaging of small proteins by CryoEM
Bacterial cell envelope homeostasis at the (post)transcriptional level
Title to be announced
Hydrologic extremes alter mechanisms and pathways of carbon export from mountainous floodplain soils
Dissecting post-transcriptional gene expression regulation in humans and viruses
Polyploidy and rediploidisation in stressful times
Prdm9 control of meiotic synapsis of homologs in intersubspecific hybrids
Title to be announced
RNA virus from museum specimens
Programmed DNA double-strand breaks during meiosis: Mechanism and evolution
Title to be announced