InterAKTions with FKBPs - mutational and pharmacological exploration

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

Kerntransport des DNA-Fragmentierungsfaktors über den klassischen Importin α/β-Transportweg

Zu den biochemischen Kennzeichen der Apoptose gehört die internukleosomale DNA-Fragmentierung, die durch den DNA-Fragmentierungsfaktor (DFF) 40 vermittelt wird. In proliferierenden Zellen wird DFF40 in Gegenwart von DFF45 exprimiert, das eine doppelte Funktion als Chaperon und Inhibitor von DFF40 erfüllt. Der entstehende DFF-Komplex wird in den Zellkern transportiert, wo DFF40 im Verlauf der Apoptose durch Spaltung von DFF45 aktiviert wird. In dieser Arbeit wird gezeigt, dass der DFF-Komplex über den klassischen Importin α/β-Transportweg in den Zellkern gelangt. Die C-terminalen Bereiche beider Untereinheiten sind für die Erkennung des DFF-Komplexes durch Importin α/β essentiell. Wenigstens je eine Gruppe basischer Aminosäuren in DFF40 (RLKRK) und DFF45 (KRAR) wird für den Transport des DFF-Komplexes in den Zellkern benötigt. Der basische Bereich KRAR ist darüber hinaus das entscheidende Element für den Kernimport des DFF45-Monomers. Weitere Experimente weisen daraufhin, dass DFF45 ein klassisches monopartites Kernlokalisationssignal (NLS) besitzt, das den Import in den Zellkern über den Importin α/β-Transportweg vermittelt. Außerdem enthält auch der C-Terminus von DFF40 ein unabhängiges NLS und aktiviertes DFF40 interagiert nicht nur mit Importin α/β sondern auch mit Importin β. Allerdings konnte der Transport von DFF40 und damit die Funktionalität dieser Interaktion nicht nachgewiesen werden. Die Tatsache, dass beide DFF-Untereinheiten NLSs enthalten spricht dafür, dass diese Signale durch unabhängige Bindung an Importin α/β den Kernimport des DFF-Komplexes bewirken. Die Interaktion zwischen Importin α/β und dem DFF-Komplex war jedoch deutlich stärker als die Bindung von Importin α/β an das DFF45-Monomer oder aktives DFF40. Weiterhin war die Importin α/β-Bindung an den DFF-Komplex stark verringert, wenn entweder der C-Terminus von DFF40 oder von DFF45 deletiert war. Diese Ergebnisse weisen auf eine Kooperativität bei der Bindung von Importin α/β hin, die durch simultane Interaktion von DFF40 und DFF45 an ein Importin α/β-Heterodimer erklärt werden kann. Weitere Ergebnisse lassen vermuten, dass diese Interaktion durch ein klassisches bipartites NLS vermittelt wird. Aufgrund der präsentierten Daten schlagen wir ein Interaktionsmodell vor, wonach die essentiellen basischen Bereiche in DFF40 (RLKRK) und DFF45 (KRAR) ein intermolekulares bipartites NLS bilden, das von Importin α/β erkannt wird

The FKBP51-Akt interaction depends on the conformation of Akt.

<p><b>A</b> HEK293T cells were transfected with FLAG-tagged FKBP51^K352A/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>B</b> GSH beads loaded with purified activated GST_Akt1ΔPH were incubated with FKBP51 with or without AMP-PNP. Eluates were analyzed by Western blotting.</p

Influence of the PH domain and the activation status of Akt.

<p><b>A</b> Domain structure of Akt <b>B</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. <b>C</b> and <b>D</b> HEK293T cells were transfected with the indicated HA-tagged Akt1 phosphorylation site mutants with or without co-transfected FLAG-tagged FKBP51^K352A/R356A (TPR_mut). After 2 days cells were collected, lysed, immunoprecipitated and analyzed by Western blotting (duplicates were analyzed for D). <b>E</b> 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.</p

Other AGC kinases can also bind to FKBP51.

<p><b>A</b> HEK293T cells were co-transfected in duplicates with GST-tagged Akt1 or ΔN_SGK^S422D and FLAG-tagged FKBP51. After 48 h, the lysates were immunoprecipitated and analyzed in duplicates by Western blotting. <b>B</b> HeLa cells were co-transfected with FLAG-tagged FKBPs and HA-tagged S6K, treated with rapamycin (25 nM) or DMSO for 60 min and lysed. Lysates were immunoprecipitated and analyzed by Western blotting.</p

Influence of FKBP inhibitors.

<p><b>A</b> Purified proteins were mixed and treated with DMSO or rapamycin (1 µM). After 3 h a GST-pull-down was performed followed by Western blotting. <b>B</b> HEK293T cells were transfected with the indicated constructs. After 48 h 1 µM FK1706 was added for 1 h. The lysates were immunoprecipitated and a Western blot was performed. <b>C and D</b> HEK293T cells were transfected with FLAG-FKBP51 and HA-tagged PHLPP1 or HA-tagged PHLPP2. After 48 hFKBP inhibitors (1 µM) or DMSO were added for 1h. Lysates were immunoprecipitated and analyzed by Western blotting. <b>E</b> HEK293T cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt phosphorylation was determined using a homogeneous time- resolved FRET assay. <b>F</b> SHSY-5Y cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt and mTOR phosphorylation was determined using a homogeneous time resolved FRET assay. <b>G</b> SU.86.86 cells were plated, treated with Gemcitabine in the absence or presence of FK1706. Cell survival relative to DMSO treated controls was determined.</p

Schematic model of possible Hsp90-Akt-FKBP51 complexes.

<p><b>A</b> FKBP51 can bind directly to Akt via its FK1 domain, but not with its FK506-binding pocket. Several other FK1-possessing FKBP homologs may bind to Akt in a similar mode. <b>B</b> Akt and several other kinases can bind to FKBP51 indirectly via Hsp90. <b>C</b> FKBP51 could assist the chaperoning of Akt by binding to Hsp90 via its TPR domain and by interacting with Akt via its FK1 domain.</p

Substrate-Selective Inhibition of Protein Kinase PDK1 by Small Compounds that Bind to the PIF-Pocket Allosteric Docking Site

International audienceThe PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery

Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase

We thank Jean-Pierre Changeux and Felix Rey for careful reading of the manuscript and support. We also thank Larissa Pietsch, Daniel Pastor-Flores, Marcelo Salierno, José Arencibia, Daniel A. Biondi, and the whole Research Group PhosphoSites for support. We thank Holger Stark and Eugen Proschak for medicinal chemistry advice. We are grateful to the ESFRI INSTRUCT Core Center Frankfurt at the Max Planck Institute of Biophysics for the use of their X-ray generators. We acknowledge synchrotron beam time at the Swiss Light Source, Paul Scherrer Institut, Villigen, and at BESSY II, Helmholtz-Zentrum Berlin, Germany. We acknowledge E-Infrastructure South (Emerald), HEC-BioSim (Archer), the PRACE Research Infrastructure (Tier-0 resources MareNostrum, Curie and Hornet, under FP7/2007-2013 grant agreement no. RI-283493), for computational resources.International audienceAllostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site and affect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins