RGS Proteins and Septins Cooperate to Promote Chemotropism by Regulating Polar Cap Mobility

Background—Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. Results—Using microfluidics and live cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G-protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. Conclusion—Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.Peer reviewe

The Dead Receptor Paradox: Insights into Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

Receptor tyrosine kinases, or RTKs, are key transducers of cellular signals in metazoans. All RTKs utilize a common architecture—an extracellular ligand binding region, a single-pass transmembrane helix, and an intracellular kinase domain—to control diverse biological processes from cellular metabolism to migration. Our understanding of how most receptors relay signals, however, remains incomplete. A particular puzzle is the collection of ~10% of RTKs that lack catalytic activity in their intracellular kinase homology domains, owing to altered amino acid residues thought necessary for phosphorylation. These so-called pseudokinases are highly evolutionarily conserved and are mutated, overexpressed, or otherwise dysregulated in certain cancers or developmental disorders. This observation serves as the basis for what I refer to herein as the dead receptor paradox; despite containing ‘dead’ kinase domains in their intracellular regions, these RTK pseudokinases are competent to bind ligands in their extracellular regions and mediate signaling. In this thesis, I ask the basic question: how do the human RTK pseudokinases detect and relay extracellular cues? Recent studies, in addition to my work described here, have led me to conclude that conformational dynamics of the pseudokinase domains is an essential component to the signaling mechanisms of these receptors. In this body of work, I demonstrate that the WNT-associated RTK pseudokinases bear close resemblance to the insulin receptor kinase domain in their structures and dynamics. I further show that RTK pseudokinases, regardless of their ability to bind nucleotides, can be targeted with small molecules to induce conformational changes. Finally, to gain insights into signaling by ‘dead receptors,’ I explored the mechanisms of WNT recognition by RTK extracellular regions. Overall, my findings support a role for pseudokinase conformational dynamics as a key step in signal transduction by these receptors, helping make sense of the dead receptor paradox and setting the stage for pseudokinase-targeted therapeutics

Systematic Analysis of Yeast F-box Proteins Reveals a New Role of Ubiquitination in Polarity Establishment

DNA is wrapped around a histone protein bundle, and these histone proteins can undergo post-translational modifications which then participate in signaling pathways to regulate important cellular functions. One such histone modification is methylation, where lysine can be mono, di, or tri-methylated. Reader proteins bind to methyl lysine and serve as docking sites for additional proteins that alter gene expression or chromatin structure. Dysregulation of histone methylation has been associated with certain types of cancer; therefore, it is necessary to understand the driving forces for binding of methylated lysine by these reader proteins to aid the development of reader protein probes or inhibitors with the desired specificity. In this project, the cation- interaction between trimethylated lysine 9 (Kme3) of H3 and the Drosophila reader protein, HP1 chromodomain, is investigated. The binding site of HP1 is comprised of three aromatic amino acids, two tyrosine residues and a tryptophan. To better understand the contribution of tryptophan in this cation-π binding event, an unnatural tryptophan analog is synthesized with 1 fluorine atom, which is highly electron-withdrawing, attached to the outer aromatic rung. Once synthesized, these unnatural analogs will then be inserted into the HP1 binding site. Comparative binding studies between the wild type and mutant protein with the Kme3 peptide will provide a better understanding of the cation-π mechanism. If Kme3 binds less tightly to the mutated binding pockets, it will indicate the importance of the tryptophan-π contribution to binding.Bachelor of Scienc