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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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Akt1 binds to several FKBPs and this interaction is direct.A HEK293T cells were transfected with HA_Akt1 and the indicated FKBP constructs. After 2 days the lysates were immunoprecipitated (IP) and analyzed by Western blotting. B Purified FKBPs and purified GST-tagged Akt1S473D (mimicking an active conformation) were subjected to a GST-pulldown followed by Western blotting. Cyclophilin, which also harbors a TPR domain, but no FK domains, was used as a negative control. (Ld = Load, E = eluates). C Purified FLAG-tagged FKBP51 and purified inactive Akt were mixed, subjected to a FLAG pulldown and analyzed by Western blotting.
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pone-0057508-g001: Akt1 binds to several FKBPs and this interaction is direct.A HEK293T cells were transfected with HA_Akt1 and the indicated FKBP constructs. After 2 days the lysates were immunoprecipitated (IP) and analyzed by Western blotting. B Purified FKBPs and purified GST-tagged Akt1S473D (mimicking an active conformation) were subjected to a GST-pulldown followed by Western blotting. Cyclophilin, which also harbors a TPR domain, but no FK domains, was used as a negative control. (Ld = Load, E = eluates). C Purified FLAG-tagged FKBP51 and purified inactive Akt were mixed, subjected to a FLAG pulldown and analyzed by Western blotting.

Mentions: Since members of the FKBP family are highly homologous to each other we asked if other FKBPs are able to bind to Akt. Surprisingly, in HEK293T cell lysates Akt1 also co-immunoprecipitated with FKBP52, FKBP25 and even with the smaller FKBP12 and 12.6, which consist only of the FK506-binding domain (Figure 1A). To examine whether these interactions are direct, we used purified GST_Akt1S473D[26], a GST-tagged constitutively active Akt mutant, as well as purified FKBP proteins [27] and performed pulldown assays (Figure. 1B). All FKBPs bound to Akt1S473D -loaded beads but not to empty beads or beads loaded with GST alone (Figure S1). No interaction was observed with purified Cyp40 [28], a closely related immunophilin, which also has a TPR domain and binds to Hsp90 but which lacks an FK506-binding domain. The direct interaction with purified FKBP51 was confirmed in a reversed pulldown using inactive untagged Akt1. Again, Akt1 was pulled down in the presence, but not the absence, of FKBP51 (Figure 1C).


InterAKTions with FKBPs--mutational and pharmacological exploration.

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

Akt1 binds to several FKBPs and this interaction is direct.A HEK293T cells were transfected with HA_Akt1 and the indicated FKBP constructs. After 2 days the lysates were immunoprecipitated (IP) and analyzed by Western blotting. B Purified FKBPs and purified GST-tagged Akt1S473D (mimicking an active conformation) were subjected to a GST-pulldown followed by Western blotting. Cyclophilin, which also harbors a TPR domain, but no FK domains, was used as a negative control. (Ld = Load, E = eluates). C Purified FLAG-tagged FKBP51 and purified inactive Akt were mixed, subjected to a FLAG pulldown and analyzed by Western blotting.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585324&req=5

pone-0057508-g001: Akt1 binds to several FKBPs and this interaction is direct.A HEK293T cells were transfected with HA_Akt1 and the indicated FKBP constructs. After 2 days the lysates were immunoprecipitated (IP) and analyzed by Western blotting. B Purified FKBPs and purified GST-tagged Akt1S473D (mimicking an active conformation) were subjected to a GST-pulldown followed by Western blotting. Cyclophilin, which also harbors a TPR domain, but no FK domains, was used as a negative control. (Ld = Load, E = eluates). C Purified FLAG-tagged FKBP51 and purified inactive Akt were mixed, subjected to a FLAG pulldown and analyzed by Western blotting.
Mentions: Since members of the FKBP family are highly homologous to each other we asked if other FKBPs are able to bind to Akt. Surprisingly, in HEK293T cell lysates Akt1 also co-immunoprecipitated with FKBP52, FKBP25 and even with the smaller FKBP12 and 12.6, which consist only of the FK506-binding domain (Figure 1A). To examine whether these interactions are direct, we used purified GST_Akt1S473D[26], a GST-tagged constitutively active Akt mutant, as well as purified FKBP proteins [27] and performed pulldown assays (Figure. 1B). All FKBPs bound to Akt1S473D -loaded beads but not to empty beads or beads loaded with GST alone (Figure S1). No interaction was observed with purified Cyp40 [28], a closely related immunophilin, which also has a TPR domain and binds to Hsp90 but which lacks an FK506-binding domain. The direct interaction with purified FKBP51 was confirmed in a reversed pulldown using inactive untagged Akt1. Again, Akt1 was pulled down in the presence, but not the absence, of FKBP51 (Figure 1C).

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