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Investigating Rac1 Subcellular Localization, Phosphorylation and Novel role in the Nucleus
- Author / Creator
- Abdrabou, Abdalla
Rac1 is a small GTPase that belongs to the Rho family. Like other Rho family GTPases, it mediates a plethora of cellular effects, including regulation of cytoarchitecture, cell size, cell adhesion, cell polarity, cell motility, proliferation, apoptosis/survival, and membrane trafficking. The Activity of Rac1 is controlled by three classes of regulatory: Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). However, accumulated evidence indicates that Rac1 activity and function are also regulated by other modifications, including RNA splicing and microRNAs, various post- translational modifications. Among various post-translational modifications, phosphorylation has been shown to play an important role in regulating the function of Rac1. Rac1 is phosphorylated at S71, Y64, and T108 by various kinases. The objective of my research is to understand how the phosphorylation of Rac1 regulates its function. I first studied the role of S71 phosphorylation in mediating Rac1 interaction with 14-3-3 proteins. Using Western blots, GST pull-down assays,
co-immunoprecipitation, and phosphorylation assays. I studied and characterized novel interaction of Rac1 protein with each of the seven 14-3-3 isoforms in response to EGF. In this thesis, I showed that that 14-3-3s interact with Rac1. Rac1 S71 mediates this interaction in both phosphorylation-dependent and -independent manners, but the phosphorylation-dependent interaction is much stronger. EGF strongly stimulates the phosphorylation of Rac1 S71 and the interaction between 14-3-3 and Rac1. Mutating S71 to A completely abolished both phosphorylation-dependent and -independent interactions between 14-3-3 and Rac1. The interaction between 14-3-3 and Rac1 mostly serves to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -γ, -σ, and -θ interact with Rac1. I then studied the effects of T108 phosphorylation on the nuclear localization and nuclear function of Rac1. I showed that in response to EGF, Rac1 was targeted to nuclear speckles (NS) and co-localized with the NS marker SRSF2. Rac1 was also partially colocalized with A’ protein of U2 snRNP (U2A’) that localizes to the actual splicing sites at the peripheral region of NS. I also showed that the NS localization of Rac1 was dependent on its T108 phosphorylation by EGF. In addition to T108 phosphorylation, Rac1 PBR and GTPase activity also contributed to its NS localization.
Moreover, Rac1 interacts with various proteins involved in pre-mRNA splicing, including SRSF2, U2A’, and hnRNPA1, as indicated by co-IP. These interactions are also dependent on T108 phosphorylation and affected by Rac1 PBR and GTPase activity. Finally, I showed that Rac1 regulated EGF-induced pre-mRNA splicing, and this is mediated by T108 phosphorylation.
- Graduation date
- Fall 2020
- Type of Item
- Master of Science
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