DOI
https://doi.org/10.25772/S6V7-VS28
Defense Date
2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Human and Molecular Genetics
First Advisor
Dr. Larisa Litovchick
Abstract
The Tuberous sclerosis complex (TSC) includes TSC1, TSC2 and the TBC1D7 subunits that together function as a principal inhibitor of the mTOR protein kinase complex 1 (mTORC1). mTORC1 is a master regulator of cell growth and proliferation that responds to signaling cues such as growth factors and nutrient availability. Proteomic studies in our lab revealed an interaction between the TSC subunits and DYRK1A, a ubiquitous protein kinase encoded by a gene located in the Down syndrome (DS) region on human chr21. In this study, we sought to validate the interaction of the TSC components with DYRK1A and to determine the functional significance of this interaction. Our analysis confirmed that DYRK1A interacts with TSC1 at the endogenous levels, and with TSC2 when overexpressed. Domain mapping of the DYRK1A-TSC interaction revealed that binding of TSC2 to DYRK1A requires TSC1. However, binding of TSC1 to DYRK1A does not require TSC2 as evidenced by the interaction observed in both WT and Null TSC2 Cells. We also observed that TSC1 is a substrate of DYRK1A kinase, and in this study, we report DYRK1A specific phosphorylation sites for the first time. Given that TSC is a major inhibitor of the mTORC1 pathway, we sought to assess the effect of DYRK1A loss on the mTORC1 activity. Interestingly, we did not observe any change in phosphorylation of mTORC1 substrates p70S6K and 4EBP1 under serum-starved conditions in both transient and CRISPR-Cas9 DYRK1A knockout (KO) cells. However, using FACS analysis, we noticed that cells lacking DYRK1A appeared smaller in size compared to controls in multiple cell lines. We also observed that global protein expression was significantly reduced in DYRK1A KO cell lines using the Puromycin-tagging Assay. Moreover, the decrease in global protein production was independent of the TSC1 protein. We next hypothesized that interaction of DYRK1A with the TSC1 complex could have an effect on the function of DYRK1A kinase. Indeed, we observed a significant decrease in DYRK1A kinase activity as evidenced by the loss of Lin52 phosphorylation at its Serine28 residue in Tsc1-null MEFs. This effect however, was found to be limited to Tsc1-null MEFs and was not replicated in various other TSC1 knockdown cell lines. However, preliminary studies suggest that TSC1 knockdown cell lines are more resistant to DYRK1A mediated cell cycle arrest as compared to controls. Overall, our study introduces DYRK1A as a novel partner of the TSC components that could mediate its non-canonical functions including mTORC1-independent regulation of cell proliferation and protein synthesis.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
4-26-2020