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SUMMARY Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a “flag” for Hsp90 dependence and stable cochaperone association.

Human epidermal growth factor receptor-3 (HER3) is a member of the type I receptor tyrosine kinase family. Several members of this family are overexpressed in various carcinomas. Specifically, HER2 is found to be overexpressed in 20 -30% of breast cancers. In contrast to epidermal growth factor receptor or HER2, the kinasedeficient HER3 self-associates readily at low nanomolar concentrations and in the absence of its ligands, various isoforms of heregulin (hrg). Binding of hrg disrupts HER3 oligomerization and leads to the formation of signaling-competent heterodimers, preferentially with HER2. Elevated levels of HER3 contribute to increased drug resistance observed in HER2-overexpressing cells. We have used the SELEX (systematic evolution of ligands by exponential enrichment) methodology to select RNA aptamers against the oligomeric state of the extracellular domains of HER3 (HER3ECD, monomeric R eceptor tyrosine kinases (RTKs) are involved in a broad spectrum of cell growth and differentiation events. RTKs are classified based on sequence homology and domain organization. Type I RTKs include the epithelial growth factor receptor (EGFR) and the human EGF receptor (HER) homologues HER2 (HER2͞neu, p185), HER3, and HER4 (also named c-erbB1-4). Overexpression of several members of this receptor family, especially EGFR and HER2, is associated with a variety of solid tumor malignancies (1-5). Overexpression of HER2 is found in 20-30% of breast cancers and results in ligandindependent activation and more aggressive growth behavior (5).Among the four mammalian type I RTKs, HER3 is unique because of its catalytically deficient kinase domain (6), its high propensity to self-associate in the absence of ligand (7), and the ability of the monomeric species of the extracellular domains (ECDs) of HER3 (HER3ECD) to assume a locked conformation, using an intramolecular tether (8). HER3 binds a variety of isoforms of the EGF homolog heregulin (hrg), and signaling relies on heterodimerization with other RTKs, preferentially HER2 (9). HER2 has a potent cytoplasmic kinase domain but is deficient in ligand binding. Simultaneous overexpression of both HER2 and HER3 is found in several cancers (10, 11), and the increased drug resistance in many HER2-overexpressing cancers depends on increased levels of HER3 or EGFR (12).Ligand-controlled signaling by type I RTKs involves receptor dimers. However, at elevated expression levels HER2 and other RTKs are likely to be engaged in a broader range of interactions. Activation of HER2 has been shown to result in the formation of large clusters of activated receptors from preexisting smaller clusters (13). For EGFR, ligand-independent interactions of receptors have been implicated in the rapid spread of signal over the entire surface of the cell after localized stimulation with immobilized ligand (14).The ECDs of RTKs provide attractive targets for macromolecular anticancer drugs. Examples include soluble ECDs of the receptors (15) and antibodies against the ECDs (16, 17). Herceptin, a humanize...

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The Escherichia coi Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations ofthe Fis crystal structure, indicates that significant deformation of DNA occurs upon Fis binding. The factor for inversion stimulation (Fis) is a small basic protein in Escherichia coli that was initially identified because of its role in stimulating site-specific DNA inversion by the Hin and Gin recombinases. In this role, Fis binds sitespecifically to a recombinational enhancer that becomes associated with the two recombination sites in a complex nucleoprotein structure. Fis has also been shown to function in phage A site-specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC-directed DNA replication (1). The x-ray crystal structure ofFis (2, 3) reveals it to be a homodimer with each 98-amino acid monomer containing four a-helices (Fig. 1) ices (D and D') are positioned only 25 A from each other, which is too short a distance to insert into adjacent major grooves of straight B-DNA. In the absence of a Fis-DNA cocrystal structure, nuclease and chemical footprinting combined with mutation data were used to construct a model of Fis bound to the distal site of the Hin gene (hin) enhancer that contains a DNA bending angle of , =60°(3, 8 §To whom reprint requests should be sent at t address. 1721The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.The ErbB or EGFR 1 family of receptors in humans includes four members, EGFR (ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4), that are involved in a wide range of differentiation and growth control events. Overexpression, especially of EGFR or ErbB2, has been observed in a variety of tumors (1-5). Controlled activation of these receptors requires binding of a ligand of the EGF or heregulin family of growth and differentiation factors, resulting in cross-phosphorylation of the dimerized receptors at specific tyrosines in the cytoplasmic portion. However, under conditions of overexpression, tyrosine phosphorylation occurs constitutively, and cells expressing elevated levels of ErbB2 show a more aggressive growth behavior (5).Of the different pairs of receptor dimers that can form, the combination of ErbB2 and ErbB3 shows the strongest potency in terms of stimulating cell proliferation (6). Many tumors that show overexpression of ErbB2, especially those that are more prone to become resistant to conventional treatment, also show elevated levels of ErbB3 (7). The ErbB2/ErbB3 dimer is unique in that ErbB2 has a potent cytoplasmic kinase domain, but its extracellular domains (ECD) fail to bind any known ligand directly (8, 9). On the other ha...

The EGFR (ERBB) family provides a model system for receptor signaling, oncogenesis, and the development of targeted therapeutics. Heterodimers of the ligand-binding-deficient ERBB2 (HER2) receptor and the kinase impaired ERBB3 (HER3) create a potent mitogenic signal, but the phosphorylation of ERBB2 in this context presents a challenge to established models of phosphorylation in trans. Higher order complexes of ERBB receptors have been observed biophysically and offer a theoretical route for ERBB2 phosphorylation, but it is not clear whether such complexes provide functionality beyond the constituent dimers. We now show that a previously selected inhibitory RNA aptamer that targets the extracellular domain (ECD) of ERBB3 acts by sterically disrupting these higher order interactions. Ligand binding, heterodimerization, phosphorylation of ERBB3, and AKT signaling are only minimally affected, whereas ERBB2 phosphorylation and MAPK signaling are selectively inhibited. The mapping of the binding site and creation of aptamer-resistant point mutants are consistent with a model of side-by-side oriented heterodimers to facilitate proxy phosphorylation, even at very low endogenous levels of receptors (below 10,000 receptors per cell). Additional modes of signaling with relevance to pathological ERBB expression states emerge at high receptor levels. Hence, higher order complexes of nonoverexpressed ERBB receptors are an integral and qualitatively distinct part of normal ERBB2/ERBB3 signaling. This mechanism of activation has implications for models of allosteric control, specificity of interactions, possible mechanisms of cross-talk, and approaches to therapeutic intervention that at present often generate experimental and clinical outcomes that do not reconcile with purely canonical, dimer-based models.oligomers | proxy activation B iochemical and structural analysis of the EGFR or ERBB (ErbB) family of receptor tyrosine kinases has provided a wealth of molecular details that have contributed significantly to our understanding of cell surface signaling and its deregulation in a broad range of diseases. The longstanding mechanistic model of receptor tyrosine phosphorylation in trans within ligand-activated dimers has undergone significant expansion in recent years. Beyond the regulation at the level of dimers, higher order clustering phenomena have been reported both for inactive and active receptor states (1-8). However, a critical and so far inaccessible question has been whether higher order complexes create qualitatively distinct signals that cannot emanate from dimers. The functional asymmetry of the closely related ERBB2/ERBB3 heterodimer presents an opportunity for experimental dissection but also a long-standing challenge to existing signaling models. ERBB2 is an orphan receptor that tyrosine phosphorylates its heterodimerization partners. ERBB3 is itself catalytically impaired but binds ligand, and its kinase domain allosterically activates its partners (9). This functional asymmetry is underscored by the fact tha...

We analyzed the propensity of the HER3 receptor and its extracellular domain (ECD) to undergo ligand-independent self-association. The HER3-ECD, purified from Drosophila S2 cells, binds the EGF-like domain of heregulin (hrg) with a K(d) of 1.9 nM as measured by surface plasmon resonance (SPR) studies. In a gel shift assay, the HER3-ECD self-associates into a uniform, slowly migrating species in a concentration-dependent manner, starting at concentrations of <10 nM. In contrast to the HER3-ECD, the ECD from the related HER2 receptor does not oligomerize under the same conditions. The direct interaction of HER3-ECDs was also demonstrated by pull-down assays and SPR measurements under physiological salt conditions. This self-association of the HER3-ECD was reversed by the addition of hrg but not by EGF. The apparent equilibrium dissociation constant for the HER3-ECD self-association is 15 nM, based on SPR measurements. In this analysis, hrg blocks HER3-ECD self-association, and the addition of hrg during the dissociation phase resulted in an accelerated off rate. This finding suggests that hrg can bind to and disrupt preexisting HER3-ECD oligomers. Full-length HER3 likewise exhibited self-association. Under conditions where co-immunoprecipitation and cross-linking of HER2 and HER3 were stimulated by hrg, HER3 self-association and cross-linking were disrupted by hrg. The implication is that the self-association of HER3-ECD favors the formation of catalytically inactive complexes of the HER3 receptor. Binding of hrg releases HER3 which may then form signaling-competent HER3-HER2 heterodimers.