Dissecting the Role of c-Src in the Regulation of Adherens Junctions using Engineered Kinases
thesisposted on 08.02.2018, 00:00 by Jennifer Klomp
Using an inducible kinase system, Rapamycin Regulated kinase (RapR-kinase), we directly activated the Src family tyrosine kinases c-Src (Src) and Lyn in human pulmonary arterial endothelial cells. We found that Src induced a transient endothelial cell barrier enhancement phase followed by a subsequent increase in permeability. Conversely, Lyn activation resulted in only disruption of the endothelial cell barrier. Similarly, we found that activation of Src but not Lyn was able to increase the rate of barrier recovery following disruption via thrombin. Further investigation revealed that Src-mediated enhancement of the endothelial cell barrier relied on accumulation of adherens junction protein VE cadherin, as well as rearrangement, and broadening of adherens junctions. In addition, we determined that phosphorylation of VE cadherin residue Y731 was required for Src-mediated endothelial cell barrier enhancement. The ability to temporally monitor the effect of direct activation of a specific kinase allowed for the identification of novel Src-mediated roles in endothelial cell barrier regulation, demonstrating the utility of using an inducible kinase system. A major limitation of the RapR-kinase system was that it only enabled activation of a kinase and not inactivation. However, due to the dynamic nature of phosphorylation, a system that allows for both specific and rapid activation and inhibition is needed for more accurate re-capitulation and dissection of signaling cascades mediated by specific kinases. Therefore, we further modified the system to allow for transient activation. By combining the RapR-kinase method with the mutation of the “gate keeper” residue in the catalytic domain and using the allele specific inhibitor 1-Naphthyl-PP1, we successfully generated a highly efficient and specific method to transiently activate kinases. Using our engineered reversible Src kinase as a model, we demonstrated that the duration of its kinase signaling can be tightly controlled in living cells. By employing this approach, we identified morphological changes induced by transient activation of Src and demonstrated the role of sequential Src-PI3K and Src-Rac1 signaling in the regulation of cell morphology. Furthermore, we expanded the technique to the Serine/Threonine kinase p38 to demonstrate its broad applicability. In summary, we described a new approach that enables transient activation of a kinase in living cells and identified new Src-dependent mechanisms for influencing changes in endothelial cell barrier function. The ability to manipulate kinase activity for a finite amount of time therefore provides a biologically relevant method of dissecting individual kinase functions and their effects on downstream signaling pathways.