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...

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding toIn rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr 36 remained bound to eIF-4E, indicating that phosphorylation of Thr 36 is insufficient for dissociation of the PHAS-I⅐eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a prolinedirected protein kinase.

The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with ␥-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine-or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.