p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.     

Effect of E(sev) and Su(Raf) Hsp83 mutants and trans-heterozygotes on bristle trait means and variation in Drosophila melanogaster

The Hsp90 protein encoded by the Hsp83 gene is required for the development of many traits in Drosophila. Hsp83 is also thought to play a role in the expression of phenotypic and genetic variability for subsequent selection and evolutionary change. Here we examine the impact of different E(sev) and Su(Raf) Hsp83 mutants on means and phenotypic variances of invariant and variable bristle traits. One of the mutants influenced the normally invariant thoracic bristle number, while none affected invariant scutellar bristle number. E(sev) alleles consistently influenced variable bristle traits while there were fewer effects of the Su(Raf) alleles. For the variable traits, none of the Hsp83 alleles had any effect on phenotypic variance, environmental variance, or developmental stability of the bristle traits. When alleles were combined in trans-heterozygotes, there were both cumulative and complementary effects on thoracic and variable bristle trait numbers, depending on the allelic combination. Overall, the results suggest that Hsp83 mutants do not have detectable effects on the phenotypic or environmental variance of bristle traits and that complementation of E(sev) and Su(Raf) Hsp83 mutants can extend to thoracic bristles as well as previously reported effects on viability. Some allelic combinations lead to more severe effects on variable bristle trait means than do single Hsp83 mutations.     

Modular Control of Cross-oligomerization: ANALYSIS OF SUPERSTABILIZED Hsp90 HOMODIMERS IN VIVO*

Homo-oligomeric proteins fulfill numerous functions in all cells. The ability to co-express subunits of these proteins that preferentially self-assemble without cross-oligomerizing provides for controlled experiments to analyze the function of mutant homo-oligomers in vivo. Hsp90 is a dimeric chaperone involved in the maturation of many kinases and steroid hormone receptors. We observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered superstabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. Superstabilized Hsp90 dimers supported robust growth of S. cerevisiae, indicating that dissociation of Hsp90 dimers could be hindered without compromising essential function. We utilized superstabilized dimers to analyze the activity of ATPase mutant homodimers in a temperature-sensitive yeast background where elevated temperature inactivated all other Hsp90 species. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of glucocorticoid receptor and v-Src, confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.     

Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.     

Nucleotide-dependent interaction of Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1, Cpr6, and Sba1

The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.     

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.     

Hsp90 phosphorylation is linked to its chaperoning function. Assembly of the reovirus cell attachment protein.

Studies on Hsp90 have mainly focused on its involvement in the activation of several families of protein kinases and of steroid hormone receptors. Little is known regarding the role of Hsp90 in the folding of nascent proteins. We previously reported that Hsp90 plays an active role in the posttranslational assembly of the C-terminal globular head of the reovirus attachment protein final sigma1. We show here that Hsp90 becomes phosphorylated in this process. However, only the unphosphorylated form of Hsp90 is complexed with final sigma1, suggesting that Hsp90 phosphorylation is coupled to the release of the chaperone from the target protein. Geldanamycin, which blocks final sigma1 maturation by preventing the release of Hsp90 from final sigma1, also inhibits Hsp90 phosphorylation. Taken together, these results demonstrate that Hsp90 phosphorylation is linked to its chaperoning function.     

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.     

Hsp90 and the quantitative variation of wing shape in Drosophila melanogaster

The molecular chaperone protein Hsp90 has been widely discussed as a candidate gene for developmental buffering. We used the methods of geometric morphometrics to analyze its effects on the variation among individuals and fluctuating asymmetry of wing shape in Drosophila melanogaster. Three different experimental approaches were used to reduce Hsp90 activity. In the first experiment, developing larvae were reared in food containing a specific inhibitor of Hsp90, geldanamycin, but neither individual variation nor fluctuating asymmetry was altered. Two further experiments generated lines of genetically identical flies carrying mutations of Hsp83, the gene encoding the Hsp90 protein, in heterozygous condition in nine different genetic backgrounds. The first of these, introducing entire chromosomes carrying either of two Hsp83 mutations, did not increase shape variation or asymmetry over a wild-type control in any of the nine genetic backgrounds. In contrast, the third experiment, in which one of these Hsp83 alleles was introgressed into the wild-type background that served as the control, induced an increase in both individual variation and fluctuating asymmetry within each of the nine genetic backgrounds. No effect of Hsp90 on the difference among lines was detected, pro,iding no evidence for cryptic genetic variation of wing shape. Overall, these results suggest that Hsp90 contributes to, but is not controlling, the buffering of phenotypic variation in wing shape.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.