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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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Activation of FAK catalytic activity initiates large dorsal ruffles via the activation of Src. (A)Rapamycin treatment of HeLa cells co-expressing RapR-FAK-YM and FRB caused formation of large dorsal ruffles. (B) HeLa cells expressing either GFP-RapR-FAK-YM (YM, 64 cells), GFP-RapR-FAK kinase-dead mutant (YM-KD, 35 cells) or GFP-tagged Y397F mutant (YM-Y397F, 47 cells) were scored for ruffle induction by rapamycin. No dorsal ruffles were seen before rapamycin addition. (C) Inhibition of Src family kinases eliminated the FAK-induced ruffles. Cells co-expressing GFP-RapR-FAK-YM and Cherry-FRB were treated with rapamycin for 1 hour and imaged before and after addition of Src family kinase inhibitor PP2. PP2 addition stopped dorsal protrusion in all cells analyzed (16 cells). (D) Activation of FAK leads to activation of Src. HeLa cells co-expressing myc-tagged Src, Cherry-FRB and either GFP-RapR-FAK-YM or its Y397F mutant were treated with rapamycin for 1 hour. Src was immunoprecipitated using anti-myc antibody and its phosphorylation on Tyr418 was assessed by immunoblotting.
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Figure 3: Activation of FAK catalytic activity initiates large dorsal ruffles via the activation of Src. (A)Rapamycin treatment of HeLa cells co-expressing RapR-FAK-YM and FRB caused formation of large dorsal ruffles. (B) HeLa cells expressing either GFP-RapR-FAK-YM (YM, 64 cells), GFP-RapR-FAK kinase-dead mutant (YM-KD, 35 cells) or GFP-tagged Y397F mutant (YM-Y397F, 47 cells) were scored for ruffle induction by rapamycin. No dorsal ruffles were seen before rapamycin addition. (C) Inhibition of Src family kinases eliminated the FAK-induced ruffles. Cells co-expressing GFP-RapR-FAK-YM and Cherry-FRB were treated with rapamycin for 1 hour and imaged before and after addition of Src family kinase inhibitor PP2. PP2 addition stopped dorsal protrusion in all cells analyzed (16 cells). (D) Activation of FAK leads to activation of Src. HeLa cells co-expressing myc-tagged Src, Cherry-FRB and either GFP-RapR-FAK-YM or its Y397F mutant were treated with rapamycin for 1 hour. Src was immunoprecipitated using anti-myc antibody and its phosphorylation on Tyr418 was assessed by immunoblotting.

Mentions: FAK is known to be overexpressed and activated in human tumors 14–16, but the specific role of its catalytic activity remains unclear. To identify processes affected specifically by FAK catalytic activity, we examined the activation of RapR-FAK-YM in HeLa cells. The Y180A/M183A mutant was used to ensure the regulation of RapR-FAK by rapamycin only, and exclude modulation by endogenous upstream factors. Consistent with previous reports showing that catalytic activity is not required for FAK’s role in growth factor-stimulated motility17, activation of RapR-FAK-YM did not significantly affect cell movement (Supplementary Fig 8). However, we did observe a distinct effect on membrane dynamics. HeLa cells normally show small peripheral ruffles that remain near the cell border. Upon addition of rapamycin, the extent of ruffling greatly increased, and very large and dynamic ruffles appeared across the dorsal surface (Fig. 3A, B, Supplementary movie S1, 36/64 analyzed cells). In control studies, cells expressing similar levels of catalytically inactive RapR-FAK-YM-KD showed no change in normal ruffling activity (Fig. 3B, 34/35 analyzed cells). RapR-FAK was localized within these ruffles (Supplementary Fig. S9). Importantly, wild-type FAK was also detected in ruffles stimulated by RapR-FAK and in those produced by PDGF treatment (Supplementary Fig. S10, S11, S12), indicating that dorsal ruffles are not an artifact of RapR-FAK mislocalization. Furthermore, FAK- fibroblasts failed to produce PDGF-stimulated dorsal ruffles (158 cells analyzed), whereas 50% of control fibroblasts expressing FAK (59 out of 118 analyzed) exhibited distinct dorsal ruffling under the same stimulation conditions. These data implicate FAK catalytic activity in the regulation of dorsal membrane protrusions.


Engineered allosteric activation of kinases in living cells.

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

Activation of FAK catalytic activity initiates large dorsal ruffles via the activation of Src. (A)Rapamycin treatment of HeLa cells co-expressing RapR-FAK-YM and FRB caused formation of large dorsal ruffles. (B) HeLa cells expressing either GFP-RapR-FAK-YM (YM, 64 cells), GFP-RapR-FAK kinase-dead mutant (YM-KD, 35 cells) or GFP-tagged Y397F mutant (YM-Y397F, 47 cells) were scored for ruffle induction by rapamycin. No dorsal ruffles were seen before rapamycin addition. (C) Inhibition of Src family kinases eliminated the FAK-induced ruffles. Cells co-expressing GFP-RapR-FAK-YM and Cherry-FRB were treated with rapamycin for 1 hour and imaged before and after addition of Src family kinase inhibitor PP2. PP2 addition stopped dorsal protrusion in all cells analyzed (16 cells). (D) Activation of FAK leads to activation of Src. HeLa cells co-expressing myc-tagged Src, Cherry-FRB and either GFP-RapR-FAK-YM or its Y397F mutant were treated with rapamycin for 1 hour. Src was immunoprecipitated using anti-myc antibody and its phosphorylation on Tyr418 was assessed by immunoblotting.
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Related In: Results  -  Collection

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Figure 3: Activation of FAK catalytic activity initiates large dorsal ruffles via the activation of Src. (A)Rapamycin treatment of HeLa cells co-expressing RapR-FAK-YM and FRB caused formation of large dorsal ruffles. (B) HeLa cells expressing either GFP-RapR-FAK-YM (YM, 64 cells), GFP-RapR-FAK kinase-dead mutant (YM-KD, 35 cells) or GFP-tagged Y397F mutant (YM-Y397F, 47 cells) were scored for ruffle induction by rapamycin. No dorsal ruffles were seen before rapamycin addition. (C) Inhibition of Src family kinases eliminated the FAK-induced ruffles. Cells co-expressing GFP-RapR-FAK-YM and Cherry-FRB were treated with rapamycin for 1 hour and imaged before and after addition of Src family kinase inhibitor PP2. PP2 addition stopped dorsal protrusion in all cells analyzed (16 cells). (D) Activation of FAK leads to activation of Src. HeLa cells co-expressing myc-tagged Src, Cherry-FRB and either GFP-RapR-FAK-YM or its Y397F mutant were treated with rapamycin for 1 hour. Src was immunoprecipitated using anti-myc antibody and its phosphorylation on Tyr418 was assessed by immunoblotting.
Mentions: FAK is known to be overexpressed and activated in human tumors 14–16, but the specific role of its catalytic activity remains unclear. To identify processes affected specifically by FAK catalytic activity, we examined the activation of RapR-FAK-YM in HeLa cells. The Y180A/M183A mutant was used to ensure the regulation of RapR-FAK by rapamycin only, and exclude modulation by endogenous upstream factors. Consistent with previous reports showing that catalytic activity is not required for FAK’s role in growth factor-stimulated motility17, activation of RapR-FAK-YM did not significantly affect cell movement (Supplementary Fig 8). However, we did observe a distinct effect on membrane dynamics. HeLa cells normally show small peripheral ruffles that remain near the cell border. Upon addition of rapamycin, the extent of ruffling greatly increased, and very large and dynamic ruffles appeared across the dorsal surface (Fig. 3A, B, Supplementary movie S1, 36/64 analyzed cells). In control studies, cells expressing similar levels of catalytically inactive RapR-FAK-YM-KD showed no change in normal ruffling activity (Fig. 3B, 34/35 analyzed cells). RapR-FAK was localized within these ruffles (Supplementary Fig. S9). Importantly, wild-type FAK was also detected in ruffles stimulated by RapR-FAK and in those produced by PDGF treatment (Supplementary Fig. S10, S11, S12), indicating that dorsal ruffles are not an artifact of RapR-FAK mislocalization. Furthermore, FAK- fibroblasts failed to produce PDGF-stimulated dorsal ruffles (158 cells analyzed), whereas 50% of control fibroblasts expressing FAK (59 out of 118 analyzed) exhibited distinct dorsal ruffling under the same stimulation conditions. These data implicate FAK catalytic activity in the regulation of dorsal membrane protrusions.

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