Date of Award

11-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

John D. Schuetz, PhD

Committee

Wei Li, PhD; Stacey Ogden, PhD; Junmin Peng, PhD; Martine Roussel, PhD; Jiyang Yu, PhD

Keywords

Scrib; SHH pathway; SHH-MB; Abcc4; Gli2 phosphorylation

Abstract

Our current knowledge of regulators in vertebrates’ Hedgehog (HH) pathway is incomplete. Gaining a better understanding of HH pathway components is appealing due to the key role of the Sonic Hedgehog (SHH) pathway in modulating the developmental proliferation of granule neuron progenitors (GNPs) in the cerebellum. Aberrant activation of SHH pathway in GNPs promotes the formation of SHH-Medulloblastoma (SHH-MB), a common pediatric brain tumor. Though SHH-MB is genetically understood, current therapies are ineffective. Recently, we identified a scaffold protein, Scribble (Scrib), as a novel interactor of our previously identified SHH pathway regulator—Abcc4. Scrib’s reported expression in the brain also suggested that Scrib had a potential connection to the SHH pathway. However, so far it is unknown whether Scrib regulates the SHH pathway. In this study, I first provided evidence that Scrib is expressed in the developing cerebellum most prominently when the HH pathway is most active. Scrib's enrichment in proliferating GNPs and purified SHH-MB tumor cells led to my hypothesis that Scrib contributes to SHH pathway activation. This hypothesis was further supported by both in vitro and in vivo studies using preclinical SHH-MB models. These studies showed that suppression of Scrib diminished the intensity of the SHH pathway and reduced tumor growth. I then used the NIH-3T3 cell line, a model system that robustly responds to SHH ligand, to elucidate Scrib's role in the SHH pathway regulation. Surprisingly, in an Abcc4-independent fashion, Scrib potently modulated the SHH pathway by affecting SHH-induced amount of nuclear Gli2, a transcription factor which is a downstream activator of the SHH pathway cascade. Next, to determine why nuclear Gli2 expression is affected, a phospho-proteomic study was conducted. This study found that Scrib influences the phosphorylation at multiple Gli2 sites. Follow-up investigations, including site-directed mutagenesis and subcellular fractionation, showed that phosphorylation of S232 is critical to both optimal Gli2 activity and nuclear Gli2 expression. These findings narrowed down the Scrib effect to Gli2 phosphorylation at a single residue —S232. In addition, I extended these studies to identify the kinase mediating the phosphorylation of Gli2 at S232. Among the candidate kinases screened, CK1ε was the most likely kinase that phosphorylates Gli2-S232.

ORCID

https://orcid.org/0009-0009-3615-6019

DOI

10.21007/aetd.cghs.2024.0012

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