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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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Related in: MedlinePlus

Mapping of the FKBP51 domains interacting with Akt.A Left: Overview of the constructs used to map the FKBP51/Akt interaction. Right: Summary of the Akt interactions of FKBP domains investigated in HEK293T cells by immunoprecipitation or by pulldown with purified proteins. (WT = wildtype). B and C HEK293T cells were transfected with the indicated constructs. After 48 h, lysates were prepared and HA- or FLAG-immunoprecipitation was performed, followed by Western blotting. D GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51 mutants for 3 h. After elution a Western blot analysis was performed. E GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51FK1 constructs for 3 h. After elution a Western blot analysis was performed and the detected with an antibody which detects the FK1 domain of FKBP51.
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pone-0057508-g005: Mapping of the FKBP51 domains interacting with Akt.A Left: Overview of the constructs used to map the FKBP51/Akt interaction. Right: Summary of the Akt interactions of FKBP domains investigated in HEK293T cells by immunoprecipitation or by pulldown with purified proteins. (WT = wildtype). B and C HEK293T cells were transfected with the indicated constructs. After 48 h, lysates were prepared and HA- or FLAG-immunoprecipitation was performed, followed by Western blotting. D GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51 mutants for 3 h. After elution a Western blot analysis was performed. E GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51FK1 constructs for 3 h. After elution a Western blot analysis was performed and the detected with an antibody which detects the FK1 domain of FKBP51.

Mentions: We next aimed to map the domains of FKBP51 that interact with Akt. First we truncated the FK506-binding domain (ΔFK1) and the FK1-like domain (ΔFK1FK2) (Figure 5A). Both deletion constructs co-immunoprecipitated with overexpressed Akt1 (Figure 5B). We also co-expressed Akt1 with two FKBP51 mutants where the PPIase activity of the FK1 domain or the Hsp90-binding capacity of the TPR domain was abolished. We also tested a construct lacking the putative C-terminal calmodulin- binding site and the isolated FK506-binding domain (FK1). In all cases, Akt1 co-immunoprecipitated with the FKBP51 constructs, although with slightly reduced efficiency for the mutants (Figure 5C). To confirm the capacity of multiple domains of FKBP51 to interact with Akt we performed pulldown assays using purified proteins (Figure 5D). The functionality of the FKBP51 proteins (with exception of the FKBP51 ΔFK1 construct) was verified by an active site titration for the FK506-binding pocket [27]. Again, all FKBP51 constructs were retained by Akt1 to a similar extent. The independence of the PPIase activity was further confirmed using a pulldown assay with the isolated FK506-binding domain of FKBP51 together with the corresponding PPIase-deficient mutant. Both proteins bound to Akt to a similar extent (Figure 5E).


InterAKTions with FKBPs--mutational and pharmacological exploration.

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

Mapping of the FKBP51 domains interacting with Akt.A Left: Overview of the constructs used to map the FKBP51/Akt interaction. Right: Summary of the Akt interactions of FKBP domains investigated in HEK293T cells by immunoprecipitation or by pulldown with purified proteins. (WT = wildtype). B and C HEK293T cells were transfected with the indicated constructs. After 48 h, lysates were prepared and HA- or FLAG-immunoprecipitation was performed, followed by Western blotting. D GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51 mutants for 3 h. After elution a Western blot analysis was performed. E GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51FK1 constructs for 3 h. After elution a Western blot analysis was performed and the detected with an antibody which detects the FK1 domain of FKBP51.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057508-g005: Mapping of the FKBP51 domains interacting with Akt.A Left: Overview of the constructs used to map the FKBP51/Akt interaction. Right: Summary of the Akt interactions of FKBP domains investigated in HEK293T cells by immunoprecipitation or by pulldown with purified proteins. (WT = wildtype). B and C HEK293T cells were transfected with the indicated constructs. After 48 h, lysates were prepared and HA- or FLAG-immunoprecipitation was performed, followed by Western blotting. D GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51 mutants for 3 h. After elution a Western blot analysis was performed. E GSH beads loaded with purified GST-tagged Akt1S473D were incubated with the indicated FLAG-tagged FKBP51FK1 constructs for 3 h. After elution a Western blot analysis was performed and the detected with an antibody which detects the FK1 domain of FKBP51.
Mentions: We next aimed to map the domains of FKBP51 that interact with Akt. First we truncated the FK506-binding domain (ΔFK1) and the FK1-like domain (ΔFK1FK2) (Figure 5A). Both deletion constructs co-immunoprecipitated with overexpressed Akt1 (Figure 5B). We also co-expressed Akt1 with two FKBP51 mutants where the PPIase activity of the FK1 domain or the Hsp90-binding capacity of the TPR domain was abolished. We also tested a construct lacking the putative C-terminal calmodulin- binding site and the isolated FK506-binding domain (FK1). In all cases, Akt1 co-immunoprecipitated with the FKBP51 constructs, although with slightly reduced efficiency for the mutants (Figure 5C). To confirm the capacity of multiple domains of FKBP51 to interact with Akt we performed pulldown assays using purified proteins (Figure 5D). The functionality of the FKBP51 proteins (with exception of the FKBP51 ΔFK1 construct) was verified by an active site titration for the FK506-binding pocket [27]. Again, all FKBP51 constructs were retained by Akt1 to a similar extent. The independence of the PPIase activity was further confirmed using a pulldown assay with the isolated FK506-binding domain of FKBP51 together with the corresponding PPIase-deficient mutant. Both proteins bound to Akt to a similar extent (Figure 5E).

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