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 signalregulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37⅐Hsp90 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.The human genome encodes up to 500 kinases, which represent one of the largest families of genes in eukaryotes (1, 2). They regulate essential intracellular processes like proliferation, differentiation, development, stress response, and apoptosis. All protein kinases share a conserved catalytic domain, which switches between an active and an inactive state (3,4). A large number of these protein kinases are dependent on the Hsp90 chaperone system, which modulates their maturation and prevents their degradation (5-8). So far only little structural information of the chaperone-kinase complex is available (9). To facilitate kinase processing by the Hsp90 chaperone machinery, the specific cochaperone Cdc37 is required (9 -11).A well described function of this cochaperone is to slow down the ATPase activity of Hsp90 (12). It is widely expected that Hsp90 and its partner protein Cdc37 bind to the catalytic domain of the kinase (13-16). It remains elusive how the ternary complex consisting of protein kinase, Hsp90 and Cdc37 is structurally organized and how the protein kinase is processed by the chaperone system. Recently Cdc37 was observed to compete with nucleotide binding to the kinase domain of B-Raf (17), but the role of Hsp90 in these structures remains enigmatic. Although Hsp90 is essential for ligand binding in case of steroid hormone receptors the role for B-Raf activation is rather unclear (18). B-Raf is mutated in many human cancers and is known to form complexes with Hsp90 and Cdc37 in cell free systems (19). The interaction with the kinase is also weakened in vivo in presence of an anticancer drug that disrupts nucleotide binding to Hsp90 (20). Given the recent evolution of Hsp90 as a potential cancer target (21-23), it is important to clarify the mechanis...

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Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.