Talented all-rounders: Fluorescence polarisation assays were developed for members of the FK506-binding protein family by using fluorescent rapamycin analogues (demonstrated in the figure). These tracers retain medium to high affinity to all tested proteins (FKBP12, -12.6, -13, -25, -51, -52). They can be used for active-site titrations, competition assays with unlabelled ligands and enable a robust, miniaturized assay adequate for high-throughput screening.FK506-binding proteins (FKBPs) convey the immunosuppressive action of FK506 and rapamycin and mediate the neuroprotective properties of these compounds, and participate in the regulation of calcium channels. In addition, the larger homologues FKBP51 and FKBP52 act as cochaperones for Hsp90 and regulate the transactivational activity of steroid hormone receptors. To further characterize these FKBPs, we have synthesized fluorescein-coupled rapamycin analogues. In fluorescence polarization assays one of these compounds retained high affinity to all tested proteins (K(d): 0.1-20 nM) and could be used for active-site titrations. To adapt the fluorescence polarization assay for high-throughput purposes, a simplified rapamycin derivative was synthesized and labelled with fluorescein. This probe showed moderate affinity for the FK1 domains of FKBP51 (177 nM) and FKBP52 (469 nM) and allowed a highly robust, optimized, miniaturized assay (Z'>0.7) sufficient for high-throughput screening of large compound libraries.

The immunosuppressant and anticancer drug rapamycin works by inducing inhibitory protein complexes with the kinase mTOR, an important regulator of growth and proliferation. The obligatory accessory partner of rapamycin is believed to be FK506-binding protein 12 (FKBP12). Here we show that rapamycin complexes of larger FKBP family members can tightly bind to mTOR and potently inhibit its kinase activity. Cocrystal structures with FKBP51 and FKBP52 reveal the modified molecular binding mode of these alternative ternary complexes in detail. In cellular model systems, FKBP12 can be functionally replaced by larger FKBPs. When the rapamycin dosage is limiting, mTOR inhibition of S6K phosphorylation can be enhanced by FKBP51 overexpression in mammalian cells, whereas FKBP12 is dispensable. FKBP51 could also enable the rapamycin-induced hyperphosphorylation of Akt, which depended on higher FKBP levels than rapamycin-induced inhibition of S6K phosphorylation. These insights provide a mechanistic rationale for preferential mTOR inhibition in specific cell or tissue types by engaging specific FKBP homologs.

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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