Hsp90 regulates p50(cdc37) function during the biogenesis of the activeconformation of the heme-regulated eIF2 alpha kinase.

Recent studies indicate that p50(cdc37) facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50(cdc37) is required for the biogenesis of the heme-regulated eIF2 alpha kinase (HRI) in reticulocyte lysate. p50(cdc37) interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50(cdc37) stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50(cdc37) function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50(cdc37) interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50(cdc37) interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50(cdc37) to bind HRI and promote its activation, but did not disrupt the native association of p50(cdc37) with Hsp90. A C-terminal truncated mutant of p50(cdc37) inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50(cdc37) were detected in cultured K562 erythroleukemia cells. These results suggest that p50(cdc37) provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.

p50(cdc37) is a nonexclusive Hsp90 cohort which participates intimately in Hsp90-mediated folding of immature kinase molecules.

Hsp90 and p50(cdc37) provide a poorly understood biochemical function essential to certain protein kinases, and recent models describe p50(cdc37) as an exclusive hsp90 cohort which links hsp90 machinery to client kinases. We describe here the recovery of p50(cdc37) in immunoadsorptions directed against the hsp90 cohorts FKBP52, cyp40, p60HOP, hsp70, and p23. Additionally, monoclonal antibodies against FKBP52 coadsorb maturation intermediates of the hsp90-dependent kinases p56(lck) and HRI, and the presence of these maturation intermediates significantly increases the representation of p50(cdc37) and hsp90 on FKPB52 machinery. Although the native heterocomplex between hsp90 and p50(cdc37) is salt-labile, their dynamic interactions with kinase substrates produce kinase-chaperone heterocomplexes which are highly salt-resistant. The hsp90 inhibitor geldanamycin does not directly disrupt the native association of hsp90 with p50(cdc37) per se, but does result in the formation of salt-labile hsp90-kinase heterocomplexes which lack the p50(cdc37) cohort. We conclude that p50(cdc37) does not simply serve as a passive structural bridge between hsp90 and its kinase substrates; instead, p50(cdc37) is a nonexclusive hsp90 cohort which responds to hsp90's nucleotide-regulated conformational switching during the generation of high-affinity interactions within the hsp90-kinase-p50(cdc37) heterocomplex.

Effects of geldanamycin, a heat-shock protein 90-binding agent, on T cell function and T cell nonreceptor protein tyrosine kinases.

The benzoquinoid ansamycins geldanamycin (GA), herbimycin, and their derivatives are emerging as novel therapeutic agents that act by inhibiting the 90-kDa heat-shock protein hsp90. We report that GA inhibits the proliferation of mitogen-activated T cells. GA is actively toxic to both resting and activated T cells; activated T cells appear to be especially vulnerable. The mechanism by which GA acts is reflected by its effects on an essential hsp90-dependent protein, the T cell-specific nonreceptor tyrosine kinase lck. GA treatment depletes lck levels in cultured T cells by a kinetically slow dose-dependent process. Pulse-chase analyses indicate that GA induces the very rapid degradation of newly synthesized lck molecules. GA also induces a slower degradation of mature lck populations. These results correlate with global losses in protein tyrosine kinase activity and an inability to respond to TCR stimuli, but the activity of mature lck is not immediately compromised. Although the specific proteasome inhibitor lactacystin provides marginal protection against GA-induced lck depletion, proteasome inhibition also induces changes in lck detergent solubility independent of GA application. There is no other evidence for the involvement of the proteosome. Lysosome inhibition provides quantitatively superior protection against degradation. These results indicate that pharmacologic inhibition of hsp90 chaperone function may represent a novel immunosuppressant strategy, and elaborate on the appropriate context in which to interpret losses of lck as a reporter for the pharmacology of GA in whole organisms.

Arthropod venom citrate inhibits phospholipase A2.

Citrate has been identified as a major component of honey bee (Apis mellifera) venom by gas liquid chromatography-mass spectrometry. A citrate concentration of 9% was found for dried bee venom by a coupled enzyme assay, aconitase-isocitric dehydrogenase. A liquid honey bee venom would contain 140 mM citrate concentration (if the solids content were 30%). Bee venom phospholipase was inhibited at a 43% level with a citrate concentration of 20 mM and calcium ion at 3 mM with the enzyme assay. Citrate was also found in the venoms of bumble bee, Bombus fervidus, 7%; yellow jacket, Vespula maculifrons, 4%; scorpion, Centruroides sculpturatus, 8%; tarantula, Grammastola cala, 8% and brown recluse spider venom gland extract, Loxoceles reclusa, 1.5% based on dried venom solids. Citrate may serve as an endogenous inhibitor of divalent metal ion-dependent enzymes in arthropod venoms as described by Francis et al. (1992, Toxicon 30, 1239-1246). Many arthropod venoms contain calcium-dependent phospholipases. A direct effect of citrate as a venom component may be possible. The presence of citrate in venoms must be considered in research on receptors, ion channels and divalent ion-dependent toxins.