Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Beverly, Levi

Committee Member

Siskind, Leah

Committee Member

Ceresa, Brian

Committee Member

Clark, Geoff

Committee Member

Roman, Jesse

Author's Keywords

UBQLN; ubiquilin; BCLb; IGF1R; EGFR; ESYT2

Abstract

There are 5 Ubiquilin proteins (UBQLN1-4, UBQLN-L), which are evolutionarily conserved and structurally similar. UBQLN proteins have 3 functional domains: N-terminal ubiquitin-like domain (UBL), C-terminal ubiquitin-associated domain (UBA) and STI chaperone-like regions in the middle. Alterations in UBQLN1 gene have been detected in a variety of disorders including Alzheimer’s disease, Amyotropic Lateral Sclerosis and lung cancer. UBQLN1 has been largely studied in neurodegenerative disorders in the context of protein quality control. Several studies have hypothesized that the UBA domain of UBQLN1 binds to poly-ubiquitin chains of substrate and shuttles it to the proteasome via its UBL domain for degradation. UBQLN1 can both facilitate degradation (Ataxin3, EPS15) and stabilize (PSEN1/2, BCLb) substrates it binds. The signal that determines this fate is unknown and there is conflicting data to support the existing working model of UBQLN1. BCLb is a member of BCL2 family of proteins that maintain the balance between apoptosis and survival in cells. BCLb is anti-apoptotic and interacts with UBQLN1. Using BCLb as a model substrate, we characterized UBQLN1-substrate interaction. We identified the first two STI domains of UBQLN1 as critical for binding of the UBA domain to these proteins slows their degradation. Similarly, we showed that UBQLN1 interacts with IGF1R and ESYT2 through the STI domains and binding stabilizes these proteins through its UBA domain. Interactions that are not dependent on STI domains, for example UBL mediated interaction with PSMD4 and BAG6, do not appear to be stabilized by UBQLN1. We conclude that fate of substrates that UBQLN1 associates with, is interaction domain specific. We used data derived from UBQLN1-BCLb interactions to model how UBQLN1 regulates IGF1R in lung adenocarcinoma cells. We have identified Ubiquilin1 as a novel interaction partner of IGF1R, IGF2R and Insulin Receptor. We demonstrate here that UBQLN1 regulates expression and activity of IGF receptors. Following loss of UBQLN1 in lung adenocarcinoma cells, there is accelerated loss of IGF1R post stimulation with ligand. Despite decreased levels of total receptors, the ratio of active:total receptors is higher in cells that lack UBQLN1. We tested for differences in synthesis, degradation, trafficking, autocrine ligand production, survival, migration potential and response to chemotherapy in lung adenocarcinoma cells that have loss of UBQLN1. UBQLN1 also regulates ligand stimulated IGF2R, Insulin Receptor (INSR), Epidermal Growth Factor Receptor (EGFR). UBQLN1 deficient cells demonstrate increased survival when serum starved and stimulation of IGF pathway in these cells increased their migratory potential by 3-fold. In conclusion, UBQLN1 is essential for normal regulation of these receptor tyrosine kinases as UBQLN1 negatively regulates total receptor levels.