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InterAKTions with FKBPs--mutational and pharmacological exploration.

Fabian AK, März A, Neimanis S, Biondi RM, Kozany C, Hausch F - PLoS ONE (2013)

Bottom Line: The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases.The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51.Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

View Article: PubMed Central - PubMed

Affiliation: Research Group Chemical Genomics, Max Planck Institute of Psychiatry, Munich, Germany.

ABSTRACT
The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

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Influence of the PH domain and the activation status of Akt.A Domain structure of Akt B Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. C and D HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). E HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.
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pone-0057508-g003: Influence of the PH domain and the activation status of Akt.A Domain structure of Akt B Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. C and D HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). E HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.

Mentions: Next, we explored which domain of Akt is responsible for binding to FKBP51. Therefore, we performed pull-down assays with full length Akt and with an Akt construct lacking the PH domain (Figure 3A). Both constructs interacted identically with FKBP51 indicating that the PH domain is not necessary (Figure 3B). This is consistent with the observed interaction of FKBP51 with S6K and SGK, two kinases that lack the PH domain. The conformation and activity of Akt1 is regulated by phosphorylation at T308 and S473. To investigate the influence of these important sites we performed immunoprecipitation assays with HEK273T cell co-expressing FKBP51 (containing a TPR-mutation to exclude confounding influences of Hsp90) together with Akt1 containing a series of phosphorylation-resistant or phosphomimetic substitutions at T308 and/or S473. All these Akt constructs co-immunoprecipitated specifically with FKBP51 (TPR mutant) but not with mock-transfected controls (Figure 3C and Figure 3D). The phosphorylation status of T308 in the activation loop of Akt was not important for the interaction with FKBP51 under these cellular conditions whereas the phosphoresistant mutation S473A (lane 3 and 4) slightly increased binding of FKBP51. We next controlled the Akt activation status by stimulating or starving the cells or by inhibition of the PI3K pathway using wortmannin (Figure 3E). As expected, starvation (lane 2) and wortmannin treatment (lane 4) reduced phosphorylation of Akt at S473 and correlated with a slightly reduced binding to FKBP51. The underlying reasons for discrepancy to the results observed with the S473A mutant remain to be established. Contrary to the findings by Pei et al. [23], we observed an increase – not a reduction – in Akt S473 phosphorylation upon co-expression of FKBP51.


InterAKTions with FKBPs--mutational and pharmacological exploration.

Fabian AK, März A, Neimanis S, Biondi RM, Kozany C, Hausch F - PLoS ONE (2013)

Influence of the PH domain and the activation status of Akt.A Domain structure of Akt B Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. C and D HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). E HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585324&req=5

pone-0057508-g003: Influence of the PH domain and the activation status of Akt.A Domain structure of Akt B Purified GST-tagged active Akt or a mutant lacking the PH domain were incubated with purified FLAG-tagged FKBP51. After 3 h, the interaction was tested by GST pulldown and Western blotting. C and D HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). E HEK293T cells were starved for 16 h, stimulated with FCS for 45 min or treated with wortmannin. Controls were incubated in the presence of 10% FCS and treated with DMSO. Cells were lysed, immunoprecipitated and analyzed by Western blotting.
Mentions: Next, we explored which domain of Akt is responsible for binding to FKBP51. Therefore, we performed pull-down assays with full length Akt and with an Akt construct lacking the PH domain (Figure 3A). Both constructs interacted identically with FKBP51 indicating that the PH domain is not necessary (Figure 3B). This is consistent with the observed interaction of FKBP51 with S6K and SGK, two kinases that lack the PH domain. The conformation and activity of Akt1 is regulated by phosphorylation at T308 and S473. To investigate the influence of these important sites we performed immunoprecipitation assays with HEK273T cell co-expressing FKBP51 (containing a TPR-mutation to exclude confounding influences of Hsp90) together with Akt1 containing a series of phosphorylation-resistant or phosphomimetic substitutions at T308 and/or S473. All these Akt constructs co-immunoprecipitated specifically with FKBP51 (TPR mutant) but not with mock-transfected controls (Figure 3C and Figure 3D). The phosphorylation status of T308 in the activation loop of Akt was not important for the interaction with FKBP51 under these cellular conditions whereas the phosphoresistant mutation S473A (lane 3 and 4) slightly increased binding of FKBP51. We next controlled the Akt activation status by stimulating or starving the cells or by inhibition of the PI3K pathway using wortmannin (Figure 3E). As expected, starvation (lane 2) and wortmannin treatment (lane 4) reduced phosphorylation of Akt at S473 and correlated with a slightly reduced binding to FKBP51. The underlying reasons for discrepancy to the results observed with the S473A mutant remain to be established. Contrary to the findings by Pei et al. [23], we observed an increase – not a reduction – in Akt S473 phosphorylation upon co-expression of FKBP51.

Bottom Line: The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases.The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51.Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

View Article: PubMed Central - PubMed

Affiliation: Research Group Chemical Genomics, Max Planck Institute of Psychiatry, Munich, Germany.

ABSTRACT
The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

Show MeSH
Related in: MedlinePlus