Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Michael Hopkins - North Carolina Central University
Co-Author(s): Melissa Locke, University of Califoonia, Berkeley, CA Michael Rape, HHMI
Ubiquitin plays an essential role in virtually all cellular processes. In this study, we examined the effects of ubiquitin signaling on the cell-fate determination of human embryonic stem cells (hESC) to neuronal lineages. The Cullin-RING-ligase (CRL) family, the largest class of ubiquitin ligases, plays a critical role in human development. The CRL is a multi-subunit ligase complex that recognizes substrates through a substrate adaptor protein which determines substrate specificity for the ligase. Specifically, we are studying Cullin-RING-Ligase 3 (CUL3) which is frequently mutated in a variety of developmental diseases and mouse knockout of CUL3 is embryonic lethal. Our protein of interest Kelch 8 (KLHL8), a substrate adaptor of CUL3, is upregulated during embryoid body formation and forms an active ligase complex with CUL3. KLHL8 knockdown alters the normal hESC neuronal differentiation pathway and leads to an increase in neural crest cells and a decrease in central nervous system (CNS) precursor cells. To identify important interactors of KLHL8 we performed immunoprecipitation and mass spectrometry assays. These experiments identified TYK2 as a potential interactor of the CUL3-KLHL8 complex. We confirmed interaction, of KLHL8 and TYK2 by co-expression in HEK293T cells and cloned truncation mutations of TYK2 to identify regions important for binding. We also obtained preliminary data that TYK2 is ubiquitylated in a KLHL8 dependent manner and that KLHL8 stabilizes TYK2 protein levels. This study indicates that KLHL8 regulates TYK2 and its associated signaling pathways, and may provide insight into the cellular mechanism behind the observed phenotype of KLHL8 knockdown during neuronal stem cell differentiation.
Funder Acknowledgement(s): This research was supported by the Amgen foundation at the University of California, Berkeley, and funded by the Howard Hughes Medical Institute EXROP program.
Faculty Advisor: Tonya Gerald, firstname.lastname@example.org
Role: The prior research for my project was conducted by my mentor Melissa and other members of the lab. The research that I specifically conducted was the immunoprecipitation assay that helped suggest that Tyk2 is an interacting protein of KLHL8 and is ubiquitylated and phosphorylated in a KLHL8 and the cloning and binding assessment of TYK2 truncation mutations, which indicated that the kinase domain of Tyk2 is imperative for KLHL8 interaction.