Pharmacology with a courtesy appointment in Chemistry
909 S Wolcott St., 5097 COMRB
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Protein engineering and synthetic biology
One of the directions of my laboratory is development of novel tools for interrogation of signaling pathways in living cells. We employ protein engineering approaches to gain control of individual proteins, reconstruct and manipulate specific signaling pathways, and build artificial signaling networks. These innovative technologies allow us to overcome limitations of existing methods and tackle scientific problems from a different perspective.
An example of our previously developed tools is a novel method for activation of kinases in living cells. Using a combination of an engineered allosteric switch, the iFKBP domain, and a small membrane permeable molecule, rapamycin or its non-immunosuppresive analogs, we can now control the activity of a selected kinase with high specificity and precise temporal control. Using this method we have developed rapamycin-regulated (RapR) analogs of several kinases, representing both Tyr and Ser/Thr kinases (FAK, Src, Fyn, Yes, Pak1 and p38).(Karginov A.V. et al Nat Biotechnol. 2010, 28:743-7)
Further modification of the RapR technology allowed us to control individual signaling pathways mediated by the engineered kinase. Initiation of a specific signaling pathway is often determined by activation of a kinase at a specific subcellular location or in a specific protein complex. Efficient activation of a RapR-kinase requires interaction with an accessory protein FRB. By targeting FRB to a subcellular compartment or inserting it into a known binding partner for the RapR-kinase we were able to achieve activation of specific signaling pathways in living cells. Application of this method allowed us to determine the role of individual signaling pathways downstream of Src kinase in regulation of cell morphodynamics. (Karginov A.V. et al Nat Chem Biol. 2014, Apr;10(4):286-90).
In our most recent work, we developed an approach for transient activation of kinases. By combining two protein engineering strategies we were able to turn on and turn off a specific kinase with tight temporal control in living cells. Using this tool we were able to observe morphological changes mediated by transient activation of kinase c-SRC for various periods of time. The first wave of morphological changes, where the cell seemed to expand and then contract again, was driven by kinase activation and inactivation. Interestingly, the second wave of cell expansion occurred after the kinase was already “switched off” and this effect depended on how long the kinase was "left" on before inactivation. These experiments demonstrated that even short-term stimulation of a kinase can have a long lasting effect on cell behavior and allowed us to identify molecular mechanism driving these events. (Klomp JE, Huyot V, Ray AM, Collins KB, Malik AB, Karginov AV. Mimicking transient activation of protein kinases in living cells. Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):14976-14981)
Cell-cell interactions and cell migration.
Interactions between cells, cell adhesion to extracellular matrix, and cell migration are fundamental cell functions that are often dysregulated in human diseases. We study signaling pathways regulating these processes in endothelial and cancer cells. Our effort is directed on better understanding of molecular mechanisms controlling endothelial barrier, angiogenesis, and cancer metastasis.
One of our current projects focuses on the role of Src family kinases (SFKs) in regulation of endothelial barrier. Current prevailing model suggests that activation of SFKs leads to increased endothelial permeability. Thus, targeting of SFK-mediated signaling is proposed for potential therapeutic treatment of pathological conditions associated with increased vascular permeability. However, growing evidence indicates that SFKs may also play a critical role in restoration and enhancement of endothelial barrier. Signaling pathways downstream of SFKs that promote barrier enhancement are poorly understood. Furthermore, different members of Src family may play different roles in regulation of endothelial barrier but their specific functions still need to be determined. Our engineered tools allow us to induce activation of individual Src family members and dissect downstream signaling events controlling endothelial barrier. Elucidation of barrier enhancing and barrier disrupting pathways mediated by SFKs will aid development of better treatment for pathological dysregulation of vascular permeability.
Another direction in my laboratory is aimed at understanding the initial step of angiogenesis – degradation of extracellular matrix by endothelial cells. This process is mediated by podosomes, specialized structures that regulate localized secretion of matrix degrading enzymes. This is the key event that allows endothelial cells to invade extracellular matrix and initiate formation of new blood vessels. However, processes regulating formation of podosomes in endothelial cells are not well understood. We focus on identifying novel components of podosomes in endothelial cells and dissecting their role in regulation of angiogenesis.
- Karginov AV, Tsygankov D, Berginski M, Chu PH, Trudeau ED, Yi JJ, Gomez SM, Elston TC, Hahn KM. Dissecting motility signaling through activation of specific Src-effector complexes. Nat Chem Biol. In Press.
- Patel M, Karginov AV. Phosphorylation-mediated regulation of GEFs for RhoA. Cell Adh Migr. 8, 2013.
- Karginov AV, Hahn KM. Allosteric activation of kinases: design and application of RapR kinases. Curr Protoc Cell Biol.Chapter 14:Unit 14.13, 2011.
- Karginov AV, Zou Y, Shirvanyants D, Kota P, Dokholyan NV, Young DD, Hahn KM, Deiters A. Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J Am Chem Soc.133:420-3, 2011.
- Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM. Engineered allosteric activation of kinases in living cells. Nat Biotechnol. 28:743-7, 2010.