Nucleotide-Free sB-Raf is Preferentially Bound by Hsp90 and Cdc37 In Vitro.

The molecular chaperone Hsp90 and its cofactor Cdc37 are required for the stability of protein kinases in the cellular environment. Upon pharmacological inhibition of Hsp90, the Hsp90-dependent kinases are degraded quickly by the proteasome. Clear physiological evidence for the formation of heterooligomeric complexes between the chaperone system and its kinase clients exist, but the mechanisms of client processing are still enigmatic. Here, we investigate the interaction of the chaperone system with a stabilized fragment of the Hsp90-dependent protein kinase B-Raf (sB-Raf). sB-Raf is aggregation prone at elevated temperatures. We find that nucleotide binding strongly stabilizes the folded state of sB-Raf and suppresses its aggregation. Also, Cdc37 and Hsp90 in combination can suppress sB-Raf aggregation while forming a ternary complex with the kinase. The presence of nucleotides leads to the dissociation of the kinase from the ternary chaperone complex, implying that the stabilization of the kinase by nucleotides reduces its affinity toward the chaperone machinery. Human Cdc37-Hsp90 complexes can bind to kinase, if the NM domain of the chaperone is present. Nematode Cdc37, which does not require the N-terminal Hsp90 domain for binding, can form a ternary complex with the MC construct of Hsp90, which lacks the aggregation propensity of sB-Raf. Like the full-length complex, this interaction is sensitive to ATP binding to sB-Raf. We thus find that the interaction between sB-Raf and the Hsp90 chaperone system is based on contacts with the M domain of Hsp90, which contributes in forming the ternary complex with CeCdc37 as long as the kinase is not stabilized by nucleotide.

Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites.

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.

Cdc37 (cell division cycle 37) restricts Hsp90 (heat shock protein 90) motility by interaction with N-terminal and middle domain binding sites.

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.

A network of genes connects polyglutamine toxicity to ploidy control in yeast.

Neurodegeneration is linked to protein aggregation in several human disorders. In Huntington's disease, the length of a polyglutamine stretch in Huntingtin is correlated to neuronal death. Here we utilize a model based on glutamine stretches of 0, 30 or 56 residues in Saccharomyces cerevisiae to understand how such toxic proteins interfere with cellular physiology. A toxicity-mimicking cytostatic effect is evident from compromised colony formation upon expression of polyglutamines. Interestingly, diploid cells are insensitive to polyglutamines and haploid cells can escape cytostasis by hyperploidization. Using a genome-wide screen for genes required to obtain the cytostatic effect, we identify a network related to the budding process and cellular division. We observe a striking mislocalization of the septins Cdc10 and Shs1 in cells arrested by polyglutamines, suggesting that the septin ring may be a pivotal structure connecting polyglutamine toxicity and ploidy.

Functions of the Hsp90 chaperone system: lifting client proteins to new heights.

The molecular chaperone Hsp90 is an essential protein in eukaryotic organisms and is highly conserved throughout all kingdoms of life. It serves as a platform for the folding and maturation of many client proteins including protein kinases and steroid hormone receptors. To fulfill this task Hsp90 performs conformational changes driven by the hydrolysis of ATP. Further, it can resort to a broad set of co-chaperones, which fit the Hsp90 machinery to the needs of specific client proteins. During the last years the number of identified co-chaperones has been consistently rising, implying that the client spectrum of Hsp90 may be much more diverse and larger than currently known. Many cofactors contain a TPR-domain for interactions at the C-terminus of Hsp90 and in many cases their functions and client sets remain to be uncovered. Hsp90 is also a putative target to interfere with cancerous and infectious diseases. Thus the knowledge on more of its cellular functions would provide also more therapeutic options for the future. In this review we compile the current knowledge on the Hsp90 ATPase mechanism, cofactor regulation and prospects of Hsp90 inhibition.