Signal Responsiveness of IκB Kinases Is Determined by Cdc37-assisted Transient Interaction with Hsp90

Michael Hinz,Meike Broemer,Seda Çöl Arslan,Albrecht Otto,Eva-Christina Mueller,Rudolf Dettmer,Claus Scheidereit

doi:10.1074/jbc.m705785200

Michael Hinz, Meike Broemer + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m705785200

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Abstract

The IkappaB kinase (IKK) holocomplex, containing the kinases IKKalpha, IKKbeta, and the scaffold NEMO (NF-kappaB essential modifier), mediates activation of NF-kappaB by numerous physiological stimuli. Heat shock protein 90 (Hsp90) and the co-chaperone Cdc37 have been indicated as additional subunits, but their specific functions in signal transduction are indistinct. Using an RNA interference approach, we demonstrate that Cdc37 recruits Hsp90 to the IKK complex in a transitory manner, preferentially via IKKalpha. Binding is conferred by N-terminal as well as C-terminal residues of Cdc37. Cdc37 is essential for the maturation of de novo synthesized IKKs into enzymatically competent kinases but not for assembly of an IKK holocomplex. Mature IKKs, T-loop-phosphorylated after stimulation either by receptor-mediated signaling or upon DNA damage, further require Hsp90-Cdc37 to generate an activated state. Thus, the present data denote Hsp90-Cdc37 as a transiently acting essential regulatory component of IKK signaling.

Highlights

NOVEMBER 2, 2007 VOLUME 282 NUMBER 44 pathway induces C-terminal processing of NF-␬B2/p100 to generate p52-containing complexes [5]
Heat shock protein 90 (Hsp90)-Cdc37 Interacts with the Core IKK Complex in a Transitory Manner—To determine the role of Hsp90 and Cdc37 in IKK signaling, we analyzed the mode of their association with the IKK holocomplex
Because small amounts of Cdc37 and Hsp90 trailed into the IKK fractions, a stoichiometric contribution of Cdc37 and Hsp90 to the IKK complex could still be possible if Cdc37 and Hsp90 were present in high molar excess over the IKK components

Summary

EXPERIMENTAL PROCEDURES

HeLa and 293 cells were grown in Dulbecco’s modified Eagle’s medium (PAA Laboratories), supplemented with 10% fetal calf serum, 1. The IKK complex was precipitated with an IKK␥ antibody, and co-immunoprecipitation of Hsp and Cdc was analyzed by Western blotting. B, 293 cells were transfected with FLAG-Cdc and different Myc-IKK constructs as indicated. Cell extracts (Input) were immunoblotted to determine expression levels of IKK␣, IKK␤, and Cdc. D, 293 cells were transfected with FLAG-Cdc wild type (WT) or deletion constructs along with Myc-IKK␣. Precipitates were analyzed for Myc-IKK, Hsp, and FLAG-Cdc by Western blotting.

RESULTS

To investigate the mode of

DISCUSSION