Biochemical and single-molecule analysis of signaling molecules

Abstract

Mammalian cells use a variety of molecules to receive, process, and transmit information from the extracellular environment. Proteins receive and transmit signals through direct protein-protein interaction at the membrane surface and within the cytoplasm. Similarly, membrane lipids facilitate signal transmission across the membrane by direct interaction with intracellular proteins. In this disertation, I have investigated protein-protein and lipid-protein interactions of important signaling molecules by employing biochemical methods and novel single-molecule approaches. Mammalian target of rapamycin (mTOR) is a master regulator of mammalian cell growth and proliferation. Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. However the mechanism by which PA activates mTORC1 signaling has remained unknown. In Chapter I of my thesis, I discovered a new mechanism by which the lipid second messenger phosphatidic acid (PA) regulates mTORC1 complex. PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Additionally, PA binding to mTORC1 leads to an increase in kinase activity by an allosteric effect, independently of FKBP38. In conclusion, a dual mechanism for PA activation of mTORC1 is proposed– PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. In chapter III of my thesis, in collaboration with Dr. Ankur Jain from Dr. Taekjip Ha laboratory, I used the single-molecule pulldown (SiMPull) assay to investigate the stoichiometry and assembly of mTORCs. After validating this novel approach with mTORC1, confirming a dimeric assembly as previously reported, I show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, the data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed the distinction of complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR–raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential. Signaling phospholipids are critical mediator of biological processes such as cell growth, proliferation, and metabolism. Recognition of signaling phospholipids by proteins is important for the targeting and initiation of many signaling cascades, but the mechanisms that regulate such interactions are not completely understood. The majority of the biophysical methods used to measure these interactions are performed with pure proteins in-vitro. In Chapter IV, in collaboration with graduate student Vasudha Aggarwal from Dr. Taekjip Ha laboratory, I have developed a single-molecule fluorescence approach to analyze lipid-protein interaction using crude cell extracts. The assay is applicable to a variety of lipid-binding domains (LBDs) expressed in cell lysates, and these LBDs are specifically pulled down by their target phospholipids. The single-molecule analysis quantitatively describes the interaction of LBDs on the lipids in real-time. These allowed the distinction of assembly features and kinetics for the different LBDs uncovering novel interaction behaviors. As an extension to cellular proteins, I determined the assembly properties of protein kinase Akt. Overall, this study demonstrates the strength of our assay to investigate protein-lipid interaction mechanisms in a new light