Mitotic-dependent phosphorylation of leukemia-associated rhogef (LARG) by Cdk1
Abstract
Rho GTPases are crucial players in the regulation of actin cytoskeleton- dependent processes, including embryonic development and mitosis. Our laboratory has discovered a novel role and localization of the leukemia- associated Rho guanine-nucleotide exchange factor (LARG) during mitosis. Rho and LARG have been implicated in diseases such as cancer, cardiovascular disease, and HIV, thus highlighting the importance for further research. The purpose of this thesis was to determine the molecular mechanism and role of mitotic-dependent phosphorylation of LARG. Our experiments are primarily conducted in vitro, in HeLa cells, a human uterine cancer cell line, and human embryonic kidney (HEK293) cells, and experiments are conducted with cellular, molecular, and biochemical approaches. Using mitotic-shift assays and kinase inhibitors, we report that LARG undergoes a mitotic-dependent and Cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Cell synchronization followed by Western blot also identified that LARG is phosphorylated and dephosphorylated during mitosis and that there is a temporal feasibility of Cdk1 as a potential kinase. Furthermore, using in vitro kinase assays, we show that LARG can be directly phosphorylated by Cdk1. Using both N- and C-terminal deletion and phosphonull mutants, we demonstrate that the mitotic-dependent shift of LARG relies on phosphorylation occurring in both termini. Using custom phosphospecific antibodies, we confirm that two sites are phosphorylated during mitosis and also in a Cdk1-dependent manner. Lastly, activity assays reveal that phosphonull LARG has more GEF activity than phosphomimetic LARG. We conclude that Cdk1 phosphorylates LARG during mitosis, phosphorylation decreases the activity of LARG, and that the dephosphorylated LARG has increased activity toward RhoA. Since LARG has diverse signaling functions beyond mitosis such as in embryonic development and disease, it is important to understand the regulation of LARG.
Subject Area
Molecular biology|Cellular biology|Biochemistry
Recommended Citation
Helms, Michelle Carll, "Mitotic-dependent phosphorylation of leukemia-associated rhogef (LARG) by Cdk1" (2015). ProQuest ETD Collection - Thomas Jefferson University. AAI3705072.
https://jdc.jefferson.edu/dissertations/AAI3705072
