Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.

Molybdate Inhibits Hsp90, Induces Structural Changes in Its C-Terminal Domain, and Alters Its Interactions with Substrates

“…At physiological temperatures, these receptors require hsp90 function to assume conformations competent to bind hormone, and this competence is maintained in receptor‚hsp90 heterocomplexes stabilized by molybdate. In addition to steroid receptors, hsp90 plays an essential positive role for several hsp90-associated kinases, including p56 lck (26)(27)(28), viral p60 src (22,36), raf (37)(38)(39)(40), and the heme-regulated eIF-2R kinase (34,41,42). These kinases exhibit moderate kinase activity when isolated as complexes with hsp90; however, these activities are typically smaller than those observed for the free kinases.…”

“…To determine the effects of geldanamycin and/or molybdate on the luciferase‚hsp90 complex, luciferase (40 µg) was denatured at 41 °C for 10 min and adsorbed for 2 h at 4 °C to affinity-purified anti-luciferase antibody (4 µL) bound to goat anti-rabbit immunoresins prepared as described previously (34). Control reaction mixtures received no luciferase.…”

“…To assess the effects of geldanamycin on interactions between hsp90 and p56 lck , p56 lck was synthesized for 30 min in RRL containing geldanamycin and/or Me 2 SO 4 vehicle (5 µg/mL and 0.5% final concentrations, respectively). Complexes between p56 lck and hsp90 were co-immunoadsorbed with 5 µL of polyclonal mouse anti-p56 lck ascites fluid bound to a 15 µL bed volume of goat anti-(mouse IgG) immunoresin (34). Immunoadsorption mixtures from 70 µL of protein synthesis reaction mixtures were washed three times with TB, once with either TB (low-salt wash) or with TB500 (high-salt wash), and twice again with TB.…”

“…After drug treatments, the reaction mixtures were chilled on ice and used in geldanamycin binding assays as previously described (22). Alternatively, AC88 anti-hsp90 monoclonal mouse IgG (2.0 µg) was immobilized on goat anti-mouse immunoresins (34), and immunoresins were washed twice in TB150. These immunoresins (15 µL packed volume) were incubated with 2.0 µL aliquots of drug-treated RRL reaction mixtures for 2.5 h. After binding, immunoresins were suspended in 100 µL of TB150 and pelleted by centrifugation, and the supernatant containing unbound proteins was analyzed by SDS-PAGE and Western blotting with rabbit antibodies directed against the N-terminus of hsp90.…”

Association of Hsp90 with Cellular Src-Family Kinases in a Cell-Free System Correlates with Altered Kinase Structure and Function

“…The existence of complexes between hsp90 and viral homologs of the src family implies the existence of similar complexes between hsp90 and cellular .rrc-family kinases. Consistent with this possibility, cellular proteins such as the heme-regulated inhibitor of protein synthesis (HRI), steroid hormone receptors (SHR), and the raf kinase gene product occur in complexes with hsp90 (Tai et al, 1986;Estes et al, 1987;Kost et al, 1989;Renoir et al, 1990;Sanchez et al, 1990a,b;Smith et al, 1990;Matts et al, 1992;Stancato et al, 1993). This conclusion is also supported by the ubiquity of hsp90-complexes among viral j/r-family kinases, since the ability to form such complexes is unlikely to result from convergent evolution of viral genes (Brugge, 1986).…”