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

Abstract

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...

Structural and functional basis of protein phosphatase 5 substrate specificity

“…Previous results suggest that association of PP5 with Hsp90 chaperone complexes is accompanied by structural changes, because within a purified Hsp90-Cdc37-Cdk4 complex, the phospho-Ser13 Cdc37 substrate is inaccessible to dephosphorylation by the nonspecific λ-phosphatase but readily accessible to dephosphorylation by PP5 (8). This conclusion is further supported by recent results indicating that residues in the vicinity of the Ser13 are responsible for binding client kinases (9,22,23) where it is likely to be buried. A conformational change would, therefore, be required to allow PP5 access to its substrate.…”

“…Phospho-Ser13 Cdc37 has a more stable and compact conformation than the WT (29), in which the phosphoSer13 is accessible to dephosphorylation by both calf intestine alkaline phosphatase (29) and λ-phosphatase (8). However, at least for the kinase B-Raf, this conformation is not a requirement for either client vs. nonclient recognition (9) or subsequent association with Hsp90 as a stable complex between Hsp90, Cdc37, and B-Raf can be formed from the individual proteins in vitro in the absence of phosphorylation (22,23), although phospho-Ser13 enhances its stability (22). These observations are consistent with recent data indicating that the primary interaction of Cdc37 with B-Raf is through a distinct C-terminal domain of Cdc37 and remote from this site of phosphorylation (9,23).…”

“…However, at least for the kinase B-Raf, this conformation is not a requirement for either client vs. nonclient recognition (9) or subsequent association with Hsp90 as a stable complex between Hsp90, Cdc37, and B-Raf can be formed from the individual proteins in vitro in the absence of phosphorylation (22,23), although phospho-Ser13 enhances its stability (22). These observations are consistent with recent data indicating that the primary interaction of Cdc37 with B-Raf is through a distinct C-terminal domain of Cdc37 and remote from this site of phosphorylation (9,23). Nonetheless, our results augment the set of known, nonoverlapping residues residing in a relatively short stretch of the Cdc37 polypeptide that encompasses Ser13 responsible for interactions that regulate Cdc37 chaperone function, namely CK2 phosphorylation (11,12), client kinase recognition in vitro (9), client kinase complex formation in vivo (20), and PP5 dephosphorylation.…”

Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase

“…There is also a network of ionic interactions between Cdc37NTD and Hsp90MD, which explains previously identified salt sensitivity of Cdc37/Hsp90 binding (Fig 5B, bottom insert). Our structure thus helps explain recent data that C. elegans Cdc37 interacts with the middle domain of Hsp90(27), rather than the NTD as observed in previous human domain binding studies. Instead of assuming that C. elegans is different or that the interactions observed with isolated domains were incorrect, we propose that Cdc37/kinase first binds Hsp90 in its open conformation as seen in the crystal structure, and then rearranges to the site on the middle domain upon Hsp90 closure, as seen in our structure.…”

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy

“…Cdc37 had been shown to interact with Hsp90 during the processing of its client kinases (46,47). Our results clearly showed that disruption of the Hsp90-Cdc37 interaction by celastrol treatment during hypertrophy abrogated subsequent interactions between Hsp90 and IKK␤ in myocytes, indicating that the Hsp90-Cdc37 interaction is essential to stabilize IKK␤ prior to the formation of a ternary complex with Hsp90.…”