Multisite phosphorylation in biomolecular condensates.

ongoing

Biomolecular condensates are membraneless organelles that serve as specialized phase-separated microenvironments within which many cellular processes can occur efficiently and with precise spatial and temporal organization. As such condensates are an important target for scientific research because they can be used as regulatory mechanisms to turn cellular processes on or off. Condensates comprise flexible multivalent biomolecules with multiple binding sites. This allows them to interact with various molecules simultaneously, enabling biomolecular clustering that can lead to phase separation. Kinase phosphorylation is a critical mechanism that triggers formation and dissolution of condensates. Once formed, condensates display emergent properties, which arise due to the collective behavior of their component biomolecules. I propose to investigate the mechanism of consecutive catalysis by kinases within condensates, and how the properties of condensates affect kinase activity. I will use synthetic condensates containing a minimal model kinase substrate that strongly partitions Protein Kinase A to examine kinase reaction kinetics in vitro and in vivo. The experiments described within will elucidate whether kinase catalysis proceeds distributively or processively within condensates. The proposed research will identify the fundamental principles of kinase-dependent condensate regulation, particularly the impact of multi-site phosphorylation on the regulatory dynamics controlling condensates. Since condenstates and kinases form a mutually responsive system regulating many basic aspects of cellular physiology, understanding how they interact will provide greater knowledge of disease mechanisms. By unraveling the intricacies of these molecular interactions, I will uncover novel insights into cellular regulation that can be used to develop innovative therapeutic strategies to address current challenges in health and disease, such as cancer and neurodegenerative disorders.