Date Approved

4-2023

Document Type

Dissertation

Degree Name

PhD in Molecular Cell Biology and Neuroscience

Department

Molecular Biology

College

Graduate School of Biomedical Sciences, Virtua Health College of Medicine and Life Sciences of Rowan University

First Advisor

Katrina Cooper, PhD

Committee Member 1

Natalia Shcherbik, PhD

Committee Member 2

Dimitri Pestov, PhD

Committee Member 3

Ronald Ellis, PhD

Committee Member 4

Alicia Melendez, PhD

Subject(s)

Autophagy; Cyclin C; Cyclin-Dependent Kinase 8; Mediator Complex; Cell Physiological Phenomena

Disciplines

Biological Phenomena, Cell Phenomena, and Immunity | Cell Biology | Cellular and Molecular Physiology | Genetic Structures | Laboratory and Basic Science Research | Life Sciences | Medicine and Health Sciences | Molecular Biology | Molecular Genetics

Abstract

Cells are exposed to an enormous amount of diverse extracellular cues but have a limited arsenal of weapons for protecting and maintaining homeostasis. To overcome these restrictions, nature has engineered proteins that have multiple functions. The pleiotropy of using one protein to carry out a variety of functions allows cells to rapidly execute tailored responses to a diverse set of signals. The Cdk8 kinase module (CKM) is a conserved detachable unit of the Mediator complex predominantly known for its role in transcriptional regulation. The CKM is composed of four proteins, the scaffolding proteins Med13 and Med12, as well as the non-canonical cyclin, cyclin C, and its cognate kinase, Cdk8. Previously it has been shown that cyclin C is a multifunctional protein that performs transcriptional and stress-induced roles at the mitochondria. The localization, post-translational modifications, and different functional domains of cyclin C regulate these separate functions.

Here we show that Med13 also has dual roles in regulating stress response following nutrient depletion. In physiological conditions, Med13 works within the CKM to negatively regulate the expression of autophagy genes (ATG). Following starvation, this repression is relieved by Snx4-assisted autophagy of Med13. Moreover, we identified Ksp1 to be the autophagic receptor protein for this novel autophagy pathway. Structural analysis by others showed that Med13 has an RNA binding region. Consistent with this, we showed that once in the cytosol, Med13 localizes to ribonucleoprotein granules known as processing bodies (P-bodies) which function in mRNA silencing, decay, and storage. In addition, we show that Med13, together with Ksp1 and Snx4, are required for the autophagic degradation of conserved P-body proteins following stress. These results illustrate the day and night jobs of Med13 in response to starvation stress.

Lastly, we illustrate that the regulation of autophagy by the CKM is evolutionarily conserved. Here we show that cyclin C promotes autophagy and proteasome activity in the murine pancreatic cancer model. Collectively, these studies demonstrate the multifunctionality and conservation of the CKM in stress response.

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