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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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The FKBP51-Akt interaction depends on the conformation of Akt.A HEK293T cells were transfected with FLAG-tagged FKBP51K352A/R356A (TPR_mut) and HA-tagged Akt1. After 2 days cells were treated with 10 µM inhibitor VIII, AT7867 or DMSO for 1 h. Cell lysates and immunoprecipitates were analyzed in duplicates by Western blotting. B GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.
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pone-0057508-g004: The FKBP51-Akt interaction depends on the conformation of Akt.A HEK293T cells were transfected with FLAG-tagged FKBP51K352A/R356A (TPR_mut) and HA-tagged Akt1. After 2 days cells were treated with 10 µM inhibitor VIII, AT7867 or DMSO for 1 h. Cell lysates and immunoprecipitates were analyzed in duplicates by Western blotting. B GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.

Mentions: Since Akt activation seemed to influence the interaction with FKBP51 at least to a certain degree we next sought to control the conformation of Akt more directly using Akt conformation-specific inhibitors. We used a classical ATP-competitive inhibitor (AT7867) (Figure 4A, lane 2), which binds and stabilizes the activated ‘PH out’ conformation of Akt (PDB code 2UW9) [29] by preventing access of phosphatases [30], and the allosteric inhibitor (inhibitor VIII) (lane1), which intercalates between the PH and the kinase domain of Akt and locks the latter in a closed inactive conformation (PDB code 3O96) [31]. As expected, the ATP-competitive inhibitor (AT7867) led to Akt hyperphosphorylation [30], [32] but it did not affect the interaction with FKBP51 (Figure 4A). This was confirmed in vitro by pulldown assays using the non-hydrolyzable ATP analog AMP-PNP (Figure 4B). As described, the allosteric inhibitor completely abolished cellular Akt S473 phosphorylation [30], [33]. Interestingly, this compound substantially reduced binding of Akt to FKBP51. This suggests that in the conformation stabilized by inhibitor VIII the binding site with FKBP51 might be masked.


InterAKTions with FKBPs--mutational and pharmacological exploration.

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

The FKBP51-Akt interaction depends on the conformation of Akt.A HEK293T cells were transfected with FLAG-tagged FKBP51K352A/R356A (TPR_mut) and HA-tagged Akt1. After 2 days cells were treated with 10 µM inhibitor VIII, AT7867 or DMSO for 1 h. Cell lysates and immunoprecipitates were analyzed in duplicates by Western blotting. B GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057508-g004: The FKBP51-Akt interaction depends on the conformation of Akt.A HEK293T cells were transfected with FLAG-tagged FKBP51K352A/R356A (TPR_mut) and HA-tagged Akt1. After 2 days cells were treated with 10 µM inhibitor VIII, AT7867 or DMSO for 1 h. Cell lysates and immunoprecipitates were analyzed in duplicates by Western blotting. B GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.
Mentions: Since Akt activation seemed to influence the interaction with FKBP51 at least to a certain degree we next sought to control the conformation of Akt more directly using Akt conformation-specific inhibitors. We used a classical ATP-competitive inhibitor (AT7867) (Figure 4A, lane 2), which binds and stabilizes the activated ‘PH out’ conformation of Akt (PDB code 2UW9) [29] by preventing access of phosphatases [30], and the allosteric inhibitor (inhibitor VIII) (lane1), which intercalates between the PH and the kinase domain of Akt and locks the latter in a closed inactive conformation (PDB code 3O96) [31]. As expected, the ATP-competitive inhibitor (AT7867) led to Akt hyperphosphorylation [30], [32] but it did not affect the interaction with FKBP51 (Figure 4A). This was confirmed in vitro by pulldown assays using the non-hydrolyzable ATP analog AMP-PNP (Figure 4B). As described, the allosteric inhibitor completely abolished cellular Akt S473 phosphorylation [30], [33]. Interestingly, this compound substantially reduced binding of Akt to FKBP51. This suggests that in the conformation stabilized by inhibitor VIII the binding site with FKBP51 might be masked.

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