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Substrate Identification of an Oncogenic Kinase: Elucidating the Pathogenesis of a Rare Liver Cancer
Get PDFFibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer with limited treatment options. This cancer primarily affects adolescents and young adults. Our lab has identified a new fusion gene in this cancer called DNAJB1-PRKACA that results from a break and re-fusion in chromosome 19. This chimeric gene results in a fusion kinase that acts as the driver of this cancer. While we have shown that the kinase activity of the fusion protein is essential for transformation, it is not currently known whether the oncogenic kinase that results from this fusion event, DNAJB1- PRKACA, phosphorylates the same substrates as PRKACA, the protein product of PRKACA. While the total phosphoproteome of a cancer can implicate critical pathway changes in the tumor versus healthy tissue, it cannot provide sufficient information on what kinase is directly responsible for the phosphorylations. Knowing which proteins DNAJB1-PRKACA is directly phosphorylating in the liver could help elucidate a stepwise mechanism for understanding the pathogenesis. Furthermore, it could provide new potential therapeutic options by targeting the downstream pathways of this oncogenic kinase. In this thesis, I will first describe my work to determine a method of directly identifying proteins that are substrates of DNAJB1-PRKACA and PRKACA. I first tested an approach developed by the Shokat Lab that uses an analog-sensitive (AS) kinase in combination with a selective adenosine triphosphate (ATP) analog to identify unique substrates of a kinase. However, serious concerns of substrate specificity of the AS-kinases were raised as I developed AS versions of DNAJB1-PRKACA and PRKACA. I pivoted to a method that kills the endogenous kinase activity of a lysate using 5\u27-(4-Fluorosulfonylbenzoyl)adenosine (FSBA); kinase reactions are performed using this kinase-inactive lysate with the purified active kinase of interest and an ATP analog that has a tag on the γ-phosphate. This results in substrates with a specific thiol tag. The ATP-γ-S analog I initially used for this method was effective in visualizing kinase activity changes via western blots, but the thiol-tags were not reliably identified using MS. With the improvement of phosphopeptide enrichment methods and encouragement from the Proteomics Resource Center, a pilot experiment was designed to enrich phosphopeptides from a kinase reaction using regular ATP, FSBAtreated mouse liver lysate, and either PRKACA or DNAJB1-PRKACA. The results of this pilot experiment showed promising differences in substrate specificity between PRKACA and DNAJB1-PRKACA so I moved forward with this assay using human hepatocyte lysate instead of mouse liver lysate. In the third chapter, I will discuss the results of the assay using kinase-inactive human hepatocyte lysate in kinase reactions to determine substrate differences between three kinases: DNAJB1-PRKACA, PRKACA, and PRKACA (L206R). PRKACA (L206R) is a PRKACA variant found in adrenal tumors of patients with Cushing\u27s disease. The L206R mutation is thought to block interaction with the regulatory subunit and pathogenesis of the adrenal tumors has been accepted to be the result of constitutive activity of this mutant catalytic subunit. Recently, two papers have suggested that there is an alteration in substrate specificity between PRKACA and PRKACA (L206R). My results demonstrate that there are differences in substrates that are directly phosphorylated by each of the three catalytic subunits: PRKACA, JPRKACA, and PRKACA (L206R). Finally, I will discuss how the results of a total phosphome study of FLC patient tumor and normal samples compared against my in vitro substrate identification assay. This comparison created a more patient-relevant and focused list of direct substrates of DNAJB1-PRKACA for further study. The thesis will conclude with discussion of the implications for the pathogenesis of FLC based on the direct substrates of interest found in these experiments and future experiments
Multimodal Strategies of Host-Seeking Mosquitoes
Get PDFMosquitoes use multiple sensory modalities, including olfaction, thermosensation, and vision, to hunt human hosts and obtain a blood-meal for egg production. Any individual sensory cue is an incomplete signal of a human host, and so a mosquito must integrate multimodal sensory information before committing to approaching and biting a person. Mosquito host-seeking behavior is thus a particularly fruitful model for studying multimodal integration because of its robustness, intricacy, and public health importance. Using tethered and free flight assays, we have teased apart responses to attractive visual and thermal cues in female Aedes aegypti, the yellow fever mosquito, uncovering their contributions to host-seeking decisions and distinctions in how they modulate their responses to those cues depending on CO2, the most salient cue in human breath. We show that mosquitoes orient towards visual contrast in flight, regardless of CO2 concentration, and then sense CO2 to unlock thermotaxis towards potential hosts. Mosquitoes across their evolutionary lineage display an impressive variety of host choices, from mammals to cold-blooded frogs to leeches and earthworms, and the algorithms they use to weigh sensory host cues likely vary just as much. Our results illustrate how such weighting is performed in one species, providing a first glimpse into how general and contingent cues are integrated to produce host-seeking behavior in mosquitoes. With the rapid development of genetic and neuroscience tools in mosquitoes, we are poised to uncover the neuronal mechanisms underlying multimodal integration in these charismatic and deadly insects
4.1 Piano-Windtet
No full text2019, January 18
4.1 Piano-Windtet, piano and wind quintet: Jörg Schneider, oboe; Alexander Glücksmann, clarinet; Fritz Pahlmann, horn; Christoph Knitt, bassoon; Thomas Hoppe, piano, performed N.H. Rice: Quintet for Piano and Winds, Op .2; Walter Gieseking: Quintet for Piano, Oboe, Clarinet, Horn, and Bassoon in B–flat Major (1919); Avner Dorman: Jerusalem Mix (2007)https://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1045/thumbnail.jp
Van Kuijk Quartet
No full text2019, February 1
Van Kuijk Quartet performed Mozart String Quartet 14, K287; Poulenc “C” from Deux poems de Louis Aragon; Les chemins de l’amour ; Schubert String Quartet 14, D810https://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1043/thumbnail.jp
Decade plug resistance box
No full textDecade plug resistance box, circa 1938
Used to control the resistance of electric conductors. Made by Leeds and Northrup Company, USA
Courtesy of Alex Kogan
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1031/thumbnail.jp
Sarah J. Schlesinger Oral History. Part 6: Building career and family
No full textInterview recorded in spring 2019. Part of The Rita and Frits Markus Library Oral History project.https://digitalcommons.rockefeller.edu/sarah-schlesinger/1005/thumbnail.jp
Molecular Model of Concanavalin A, details
No full textMolecular Model of Concanavalin A, details; circa 1970s
Courtesy of George Reeke
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1073/thumbnail.jp
Center Manifold Dynamics in Randomly Coupled Oscillators and in Cochlea
Get PDFIn dynamical systems theory, a fixed point of the activity is called nonhyperbolic if the linearization of the system around the fixed point has at least one eigenvalue with zero real part. The center manifold existence theorem guarantees the local existence of an invariant subspace of the activity, known as a center manifold, around nonhyperbolic fixed points. A growing number of theoretical and experimental studies suggest that neural systems utilize dynamics on center manifolds to display complex, nonlinear behavior and to flexibly adapt to wide-ranging sensory input parameters. In this thesis, I will present two lines of research exploring nonhyperbolicity in neural dynamics
The Role and Control of WNT Signalling in an HESC Model of Human Primitive Streak
Get PDFIn amniotes, gastrulation is marked by the creation of the primitive streak (PS) and is largely controlled by WNT, BMP, and ACTIVIN/NODAL signalling. Despite detailed characterization in model organisms, the human PS and the role these pathways play in its formation and patterning remains a mystery. In this work I focused on understanding the role and control of the WNT pathway in human PS development. Due to the ethical limitations of working with human embryos, I used an in vitro human embryonic stem cell (hESC) micropatterned gastruloid system. I first showed that in the human PS there is a conserved BMP → WNT→ NODAL signalling initiation hierarchy, and that WNT is necessary and sufficient for PS formation. Next, I found that structured subpopulations of endoderm and mesoderm emerge and self-organize depending on different BMP, WNT, and ACTIVIN/NODAL levels, and that by comparison to the mouse embryo I could arrange these subpopulations along an anterior-posterior axis. With the development of a new cell tracking technique, I was also able to identify and characterize robust cell migrations from the PS region of each gastruloid that depended on which fates the cells would ultimately adopt. Putting these pieces together, I was able to derive a rudimentary first fate map of the human PS, as well as a rough picture of the BMP, WNT, and ACTIVIN/NODAL signalling gradients that determine it. One interesting and unforeseen result from this fate map was the hint of a human organizer cell fate that emerged under joint WNT and ACTIVIN/NODAL stimulation. To characterize and functionally prove this organizer\u27s existence, I devised an ex ovo cross-species transplantation strategy grafting treated gastruloids into chick embryos. The assay demonstrated that the human cells induce and contribute autonomously to a secondary axis while inducing neural fate in the host, thus fulfilling the most stringent criteria for an organizer. This work adds an important milestone to the research program begun in 1924 with the first famous organizer experiment of Hilde Mangold and Hans Spemann, and the methods I developed have opened a door to new functional explorations and tests of early human development. Having learned more about the role of WNT in determining cell fates in the gastruloid model, I next endeavoured to understand how the spatial extent and duration of the WNT signal itself was controlled. With the use of various CRISPR/Cas9 knockout lines, I discovered that DKK1 and E-CADHERIN were the two dominant factors, with E-CADHERIN transducing boundary forces to focus WNT signalling to colony border at early times, and DKK1 controlling the late WNT pattern via cell non-autonomous negative feedback. With the help of time-lapse imaging of a fluorescent reporter line and mathematical modelling, I showed that these two factors mediate a wave of WNT signalling that spreads across the tissue to be patterned, and that this wave is a generic property of a bistable system and thus likely generalizable to other instances in development. While limited by the use of hESCs, taken together my findings provide a first glimpse into the role and control of WNT signalling early on in our own, human development
The Evolving Campus exhibit, details
No full textDetails of the exhibit The Evolving Campus
Idea, design - Olga Nilova, Special Collections Librarian
Photograph by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1016/thumbnail.jp