Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the ␥-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.Heat shock protein 90 (hsp90) 1 is a cellular chaperone that participates in multiple signal transduction pathways. Recent studies have demonstrated a requirement for hsp90, or grp94, its homolog in the endoplasmic reticulum, for the proper function of 1) the mitogen-activated protein kinase pathway (1-6); 2) activity of several tyrosine kinases (Refs. 7-9 and references therein); 3) activity of several transcription factors, including p53 (10), retinoid receptors (11), steroid and aryl hydrocarbon receptors (Refs. 12 and 13 and references therein), and hypoxia-inducible factor ␣ (14); 4) activity of the cyclin-dependent kinase CDK4 (15) and the cell cycle-associated Wee1 tyrosine kinase (16); and even 5) activity of hepatitis B virus reverse transcriptase (17). Additionally, hsp90 has been shown to participate in the refolding of certain misfolded proteins (18 -20). hsp90 comprises the core of several multi-molecular chaperone complexes that interact with proteins at different stages of their maturation. The ability of hsp90 to participate in the assembly of multiple higher order chaperone complexes no doubt contributes to its involvement in diverse cellular pathways, although those factors regulating such participation remain unclear.Until recently, yeast in which hsp90 is either mutated or conditionally suppressed has served as the only means by which to study the many functions of this chaperone in the cell. The recent observation that a class of drugs known as benzoquinone ansamycins, including herbimycin A and geldanamycin (GA), specifically bind and inhibit hsp90 and grp94 has provided a new tool for functional studies of these chaperones (9, 21). Indeed, a study of structure-activity relationships has demonstrated a high correlation between the biologic effects of the benzoquinone ansamycins and their ability to bind hsp90 (22). These drugs have also been shown to possess anti-tumor activity in preclinical models, identifying the hsp90 chaperone family as a novel target for anticancer drug development (23).For these reasons, it is of much interest to characterize the drug binding site in hsp90, both to underst...

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Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein secreted by virtually all cells in a latent form. A major mechanism of regulating TGF-beta activity occurs through factors that control the processing of the latent to the biologically active form of the molecule. We have shown previously that thrombospondin 1 (TSP1), a platelet alpha-granule and extracellular matrix protein, activates latent TGF-beta via a protease- and cell-independent mechanism and have localized the TGF-beta binding/activation region to the type 1 repeats of platelet TSP1. We now report that recombinant human TSP1, but not recombinant mouse TSP2, activates latent TGF-beta. Activation was further localized to the unique sequence RFK found between the first and the second type 1 repeats of TSP1 (amino acids 412-415) by the use of synthetic peptides. A peptide with the corresponding sequence in TSP2, RIR, was inactive. In addition, a hexapeptide GGWSHW, based on a sequence present in the type 1 repeats of both TSP1 and TSP2, inhibited the activation of latent TGF-beta by TSP1. This peptide bound to 125I-active TGF-beta and inhibited interactions of TSP1 with latent TGF-beta. TSP2 also inhibited activation of latent TGF-beta by TSP1, presumably by competitively binding to TGF-beta through the WSHW sequence. These studies show that activation of latent TGF-beta is mediated by two sequences present in the type 1 repeats of TSP1, a sequence (GGWSHW) that binds active TGF-beta and potentially orients the TSP molecule and a second sequence (RFK) that activates latent TGF-beta. Peptides based on these sites have potential therapeutic applications for modulation of TGF-beta activation.

Tumour metastasis is the principal cause of death for cancer patients. We have identified the nm23 gene, for which RNA levels are reduced in tumour cells of high metastatic potential. In this report we identify the cytoplasmic and nuclear Nm23 protein, and show that it also is differentially expressed in metastatic tumour cells. We also find that the human Nm23 protein has sequence homology over the entire translated region with a recently described developmentally regulated protein in Drosophila, encoded by the abnormal wing discs (awd) gene. Mutations in awd cause abnormal tissue morphology and necrosis and widespread aberrant differentiation in Drosophila, analogous to changes in malignant progression. The metastatic state may therefore be determined by the loss of genes such as nm23/awd which normally regulate development.