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Engineered allosteric activation of kinases in living cells


Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

(a) Schematic representation of the approach used to regulate the catalytic activity of FAK. A fragment of FKBP is inserted at a position in the catalytic domain where it abrogates catalytic activity. Binding to rapamycin and FRB restores activity. (b) The truncated fragment of human FKBP12 (amino acids Thr22 through Glu108) inserted into the kinase domain. Blue and red, full-length FKBP12; red, proposed structure of the inserted fragment. The FKBP12 is shown in complex with rapamycin and FRB (cyan). (c) Immunoblot analysis of iFKBP interaction with rapamycin and FRB. Myc-tagged FKBP12 and iFKBP constructs were immunoprecipitated from cells treated for 1 h with either 200 nM rapamycin or ethanol (solvent control). Co-immunoprecipitation of co-expressed GFP-FRB was detected using anti-GFP antibody. (d) Changes in the molecular dynamics of iFKBP upon binding to rapamycin and FRB. Warmer colors and thicker backbone indicate increasing root mean square fluctuation.

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Figure 2: Development and biochemical characterization of RapR-FAK.
Figure 3: Activation of FAK catalytic activity initiates large dorsal ruffles through the activation of Src.
Figure 4: Mechanism of regulation by iFKBP; Src regulation.

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We thank J. Edwards, D. Dominguez and V. Rao for help with construction and testing of RapR-Src and RapR-p38α constructs, B. Clarke for her design of figures and are grateful to the UNC Cancer Research Fund and the National Institutes of Health for funding (GM64346 and GM057464 to K.M.H.; GM080742 and GM080742- 03S1 to N.V.D.). We acknowledge the following gifts: anti-paxillin antibodies and the construct expressing the GST-tagged N-terminal fragment of paxillin from M. Schaller, Department of Biochemistry, West Virginia University; iRap from T. Wandless, Molecular Pharmacology Department, Stanford University; the construct for myc-tagged mouse FAK from S.K. Hanks, Vanderbilt University Medical Center; the flag-tagged mouse p38α, human FKBP12 and FRB domain of human FRAP1 DNA constructs from G. Johnson, Department of Pharmacology, University of North Carolina at Chapel Hill; AP21967 compound was provided by ARIAD Pharmaceuticals.

Author information

Authors and Affiliations



A.V.K. initiated the project, developed and validated regulation of RapR-kinases and performed the studies of FAK biological function. F.D. performed molecular modeling of FKBP12 variants, RapR-FAK and RapR-Src. P.K. performed biochemical characterization of RapR-p38. N.V.D. coordinated molecular dynamics studies. K.M.H. coordinated the study and wrote the final version of the manuscript, based on contributions from all authors.

Corresponding authors

Correspondence to Nikolay V Dokholyan or Klaus M Hahn.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–19 (PDF 3729 kb)

Supplementary Movie S1

HeLa cells co-transfected with GFP-RapR-FAK-YM and mCherry-FRB were filmed for 30 min before and 60 min after addition of rapamycin. Images were taken at one minute intervals. (MOV 1015 kb)

Supplementary Movie S2

HeLa cells co-transfected with GFP-RapR-FAK-YM and mCherry-FRB were pretreated with rapamycin for 1 hour. Cells generating large dorsal protrusions were filmed for 30 min before and 30 min after addition of PP2 compound (5 μM final concentration). Images were taken at one minute intervals. (MOV 669 kb)

Supplementary Movie S3

Molecular dynamics simulations demonstrating free movement of the G-loop (red) and iFKBP insertion (magenta) within RapR-FAK catalytic domain without the binding ligands (top) and in complex with rapamycin and FRB (bottom). The simulations were performed as described in the Materials and Methods section. (MOV 2154 kb)

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Karginov, A., Ding, F., Kota, P. et al. Engineered allosteric activation of kinases in living cells. Nat Biotechnol 28, 743–747 (2010).

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