Andrei V. Karginov, Ph.D. - US grants

DESCRIPTION (provided by applicant): It remains difficult to manipulate protein kinase activity with precise timing and localization in living cells. Furthermore, targeted manipulation of kinase activity only in selected protein complexes is currently impossible for the majority of biological studies. We have recently developed a new generally applicable method for rapamycin-regulated (RapR) activation of kinases and successfully applied it to three kinases from two different classes, tyr and ser/thr kinases (FAK, Src, and p38). Here, we propose to employ RapR technology to develop new broadly applicable methods for selective regulation of highly homologous kinases in living cells, and targeted activation of kinases only when they are in specific protein complexes. We will also achieve light- mediated localized regulation of kinases using caged rapamycin. These methods will be applied to identify the roles of different Src family kinases. These highly homologous kinases serve as a good test of the specificity of the new approaches and will provide new capabilities to answer previously intractable questions. Localized activation will be used to probe the spatio-temporal regulation of pathways modulating cell protrusion and polarization.

Abstract All Projects propose to employ novel engineered reagents that enable manipulation and interrogation of individual signaling pathways with precise spatial and temporal control. As described in each Project, reagents provided by Core C will be used to define the processes that regulate the recovery of the lung endothelial barrier. Development and optimization of these tools will require significant effort as described in the Molecular Engineering Core C. The central functions of Core C will be 1) to develop molecular tools customized for each specific question in individual Projects, 2) to evaluate the new reagents and establish protocols for their application in primary human and mouse lung microvessel endothelial cells, and 3) to provide assistance with the application of the tools, troubleshooting, and analysis of the results. The intent will be to simplify application of new technologies in the Program Project and allow participants to focus on the proposed questions requiring these reagents. The specific reagents that will be developed and employed by Core C include: 1) new tools to control localization and interaction of proteins identified by each Project; 2) reagents for regulation of activity of selected proteins in living cells, and targeted activation of these proteins in specific complexes and subcellular locations; and 3) reagents for light-mediated spatio-temporal regulation of the small GTPases Rac1 and Cdc42 and other GTPases in living cells as needed. Core C will also generate and optimize reagents for expression of engineered proteins in primary endothelial cells and animal models, and develop new reagents for manipulation and interrogation of protein interactions and cell signaling as needed. The tools developed will enable manipulation of individual signaling pathways and assessment of their roles in the regulation of endothelial barrier recovery and restoration of the integrity of adherens junctions.

Aberrant signaling by protein tyrosine phosphatases (PTPases) has been identified for multiple cancers. However, dissection of PTPase-mediated signaling pathways that drive oncogenesis is limited by the capabilities of current tools. It remains difficult to manipulate PTPase activity with precise timing and specificity in living cells. Furthermore, targeted manipulation of PTPase activity only in selected protein complexes is currently impossible for the majority of biological studies. To overcome these limitations we propose to employ novel protein engineering strategy that will enable specific activation a PTPase by rapamycin or its non- immunosuppresive analogs. To demonstrate broad applicability of this method for different PTPases we propose to generate engineered PTPases Shp2, PTP1B and RPTP-?. To achieve stimulation of specific pathways downstream of a PTPase we will target activation of Shp2 to different protein complexes with known binding partners. The reagents used in this method are genetically encoded or membrane permeable, enabling ready application in many systems. This method will provide tight temporal and spatial control of PTPase activity. Tight temporal control of engineered Shp2 will allow us to determine signaling events at different time points following activation Shp2. Targeted activation of Shp2 in complexes with it binding partners Gab1 and PZR will allow us to identify signaling pathways specifically mediated by these signaling complexes. Using combination of a recently developed method for BirA fusion protein-based biotinylation (BioID) coupled with proteomics analysis we will determine changes in Shp2-associated protein interactions and accompanied changes in protein phosphorylation.

Project Summary Endothelial barrier is regulated at the level of adherens junctions (AJs), cell-cell adhesion structures mediated by the transmembrane protein VE-cadherin. Current model suggests that the tyrosine kinase c-Src functions as a negative regulator of endothelial barrier stimulating disassembly of AJs through phosphorylation of VE cadherin. However, our studies challenge this paradigm. We found that direct activation of Src using our engineered probe transiently enhances barrier function and induces formation of morphologically distinct AJs that exhibit reduced permeability. Our preliminary evidence suggests that Src-induced enhancement of endothelial barrier is mediated through signaling pathway regulating small GTPase Arf6. We also found that phosphorylation of VE cadherin on Tyr658/Tyr731 is critical for barrier strengthening by Src. Our studies also show that Src promotes formation of new contacts with extracellular matrix (focal adhesions) and stimulates interaction of focal adhesion protein p130Cas with VE cadherin. Based on these novel findings we hypothesize that Src signaling though Arf6 and VE-cadherin, and stimulation of new focal adhesions promote formation of AJs leading to strengthening of endothelial barrier. To define the role of each pathway downstream of Src, we propose to address the following questions. 1) We will determine the role of Arf6 activity in Src-stimulated formation of AJs, and identify Src-mediated pathways that activate Arf6. 2) We will define the role of Src signaling in AJs and the role of individual phosphorylation sites on VE-cadherin in formation of new AJs and enhancement of endothelial permeability. 3) We will determine the role of focal adhesions in Src-mediated enhancement of endothelial barrier and define the role of Src signaling through specific focal adhesion proteins. The timing and location of Src-mediated signaling is critical for regulation of AJs. Thus, to achieve precise temporal and spatial control of Src-mediated processes regulating AJs, we propose to employ engineered protein tools that will allow us to regulate precisely the activity of Src and phosphorylation of VE- cadherin in living cells. The level of control is unprecedented in that Src can be selectively activated and inactivated with tight temporal control and in specific subcellular locations in living cells. Importantly, Src activation can be restricted to a specific downstream targets and subcellular locations. Using these tools, we will dissect individual Src-mediated signaling pathways controlling assembly of AJs.  

Aberrant signaling by protein kinases is one of the driving forces of tumorigenesis. Transition from physiological to oncogenic processes is often triggered by changes in temporal and spatial regulation of kinases. Dissection of these events is limited by the capabilities of current tools. It remains difficult to manipulate activity of a specific kinase with precise timing and localization in living cells. To overcome current limitations we propose to develop a novel broadly applicable optogenetic tool that will allow us to regulate kinase activity in living cells using light. To control kinase activity in time and space we will engineer a novel light-sensitive allosteric switch based on fungal photoreceptor Vivid that changes conformation upon illumination with blue light. Insertion of the engineered switch at a specific site within the catalytic domain of a kinase will allow us to achieve light-mediated regulation of the activity. This will enable tightly controlled, reversible and localized regulation of a specific kinase in living cells. To demonstrate broad applicability of this tool we will use it for regulation of oncogenic protein kinases Src, Abl and PKA. To further expand application of this strategy we propose to develop light regulated PFKFB3, a structurally different kinase that phosphorylates fructose 6-phosphate to promote glucose metabolism in cancer cells. The reagents used in this method will be genetically encoded enabling ready application in many systems. Using light-mediated regulation of tyrosine kinase Src we will determine its novel role in regulation of signaling pathways that stimulate glucose metabolism during oncogenic transformation. We will employ light-controlled PFKFB3 to identify its role in localized regulation of glycolysis in different subcellular compartments of the cell and its effect on oncogenic morphodynamic changes and cell cycle.