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Engineered allosteric activation of kinases in living cells.

Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM - Nat. Biotechnol. (2010)

Bottom Line: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems.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).Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
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.

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Related in: MedlinePlus

Design and generation of RapR-FAK. (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 hour with either 200 nMrapamycin 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 (RMSF).
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Figure 1: Design and generation of RapR-FAK. (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 hour with either 200 nMrapamycin 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 (RMSF).

Mentions: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of 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 interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. 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. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases.


Engineered allosteric activation of kinases in living cells.

Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM - Nat. Biotechnol. (2010)

Design and generation of RapR-FAK. (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 hour with either 200 nMrapamycin 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 (RMSF).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2902629&req=5

Figure 1: Design and generation of RapR-FAK. (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 hour with either 200 nMrapamycin 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 (RMSF).
Mentions: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of 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 interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. 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. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases.

Bottom Line: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems.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).Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus