Date of Award
Doctor of Philosophy (PhD)
Fibrolamellar 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’-(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’s 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.
Jarmel, Melissa, "Substrate Identification of an Oncogenic Kinase: Elucidating the Pathogenesis of a Rare Liver Cancer" (2019). Student Theses and Dissertations. 525.