PMC

HSP90 Abs delay the deactivation of HSF1 in vivo. Uninjected or HSP90 MAb-injected oocytes were subjected to a heat shock at 33°C for 1 h (HS) and then allowed to recover at 18°C for the periods of time indicated, and the HSE-binding activity of HSF1 was analyzed by gel mobility shift assay. Both heat-induced and supershifted complexes are indicated as HSF1 at the left. ns, nonspecific binding activity.

GA inhibits the heat-induced transcriptional activity of HSF1. CAT activities of CMV-CAT- or hsp70-CAT-injected oocytes that were either untreated or treated with GA (10 μg/ml, 2 h) prior to incubation at 18°C (NS) or heat shock (HS) (33°C for 2 h) were compared. Each experiment was repeated at least five times with different batches of oocytes, yielding similar results. The positions of unacetylated (Cm) and acetylated (Ac) forms of [¹⁴C]chloramphenicol are indicated on the left.

Recognition of the heat shock-activated DNA-binding form of HSF1 in gel mobility shift assays by HSP90 Abs. Aliquots of nonshocked (NS) or heat-shocked (HS) oocyte extracts were mixed with HSP90 MAb or PAbs (antiserum) and incubated for 30 min prior to DNA-binding reactions with a ³²P-labeled HSE probe, and the migration of HSF1-HSE complexes was analyzed in gel mobility shift assays. Lanes in which extracts were not mixed with antibodies are indicated (−), and the final antibody dilutions in the extract incubations are indicated above the panel. Positions of the nonspecific binding activity (ns) and heat-induced (HSF1) and supershifted (HSF1 + ab) complexes are indicated.

Microinjected HSP90 Abs inhibit the heat-induced transcriptional activity of HSF1. (A) CAT assay of oocytes microinjected with hsp70-CAT (left) or CMV-CAT (right). Oocytes were injected with HSP90 MAb 1 h before heat shock (33°C for 1 h) treatment (HS), and the expression from each promoter was compared directly to that in similarly treated oocytes without injected Ab. Each experiment was repeated at least five times with different batches of oocytes, yielding similar results. The positions of unacetylated (Cm) and acetylated (Ac) forms of [¹⁴C,]chloramphenicol are indicated on the left of each panel. Background CAT activity of non-plasmid-injected oocytes is shown in the right panel (cont). NS, no heat shock. (B) CAT assay of oocytes microinjected with reporters as described above. HSF1 PAbs were injected into oocyte nuclei essentially as described above for HSP90 Ab.

(GA) enhances the heat-inducible HSE-binding activity of HSF1 and delays deactivation during recovery. (A) Oocytes were incubated for 2 h in the presence of 10 μg of GA (G) per ml or in DMSO (D) or left untreated (U) prior to heat shock at 30°C for the times indicated. The HSE-binding activities of HSF1 were then compared by gel mobility shift assay. (B) Gel mobility shift assay of oocytes that were treated with GA or DMSO as described above, heat shocked at 33°C for 1 h and then allowed to recover at 18°C for the periods of time indicated. The positions of activated HSF1 and nonspecific (ns) bands are indicated at the left of each panel.

Identification of HSP90-HSF1 heterocomplexes in Xenopus oocyte nuclei. (A) Immunoblot showing subcellular distribution of HSP90. Oocytes were incubated at a nonshock (NS) temperature (18°C) or heat shocked (HS) at 33°C for 2 h, and proteins from single intact oocytes or from the nuclear and cytoplasmic fractions were separated by SDS-PAGE along with purified bovine HSP90 as a control. HSP90 was detected with an HSP90 PAb. The positions of molecular mass standards are shown on the left. (B) Coimmunoprecipitation of HSF1 and HSP90 from oocyte nuclei. HSF1 and YY1 were immunoprecipitated with rabbit anti-mouse HSF1 PAb () or YY1 PAb (Santa Cruz). Nuclear extracts were isolated from nonshocked or heat-shocked (33°C for 1 h) oocytes. Immunoprecipitated material was subjected to SDS-PAGE and transferred to nitrocellulose, and the blot was probed with HSP90 and HSF1 Abs as indicated.

Model of HSF1 regulation by HSP90. Our data indicate that HSP90 interacts with the inactive monomeric and active trimeric forms of HSF1 and could be involved in regulating the interconversion between these forms (A and C). Both HSP90 Abs and GA delay trimer disassembly and inhibit heat-induced transcription of HSF1 (D). These observations suggest a role for HSP90 in modulation of the transcriptional activation domain (B) or that HSP90 dissociation from trimers is required for transcriptional competence. We also hypothesize that HSP90 could link HSF1 to cellular signaling molecules (B). Details of HSF1 structure are not shown, although the shape change from a circle to an ellipse is intended to indicate conformational changes associated with trimerization. No data are presented for the one-to-one stoichiometric relationship between HSP90 and HSF1, which is shown only for simplicity. Although HSP70 is not represented here, we do not imply its absence of involvement in any of these processes.

HSP90 interacts with HSF1 in vivo. (A) Immunoblot of nonshocked (NS) and heat-shocked (33°C, 2 h) oocytes that were either uninjected or microinjected with HSP90 MAb. HSP90 was detected with HSP90 primary Ab, and injected HSP90 Ab was detected with the secondary antibody (as described in Materials and Methods). Endogenous oocyte HSP90 and injected HSP90 MAbs present in extracts are indicated on the right. The positions of molecular mass standards are indicated on the left. (B) The migration of HSF1-HSE complexes was analyzed by gel mobility shift assay of uninjected or HSP90 MAb-injected oocytes that were not shocked (NS) or heat shocked (HS) at 33°C for the times indicated. The heat-activated HSF-HSE (HSF1) and HSP90 Ab-supershifted (HSF1+ab) complexes are indicated on the right. ns, nonspecific binding activity. (C) An experiment similar to that for panel B was performed with HSP90 antiserum (pAb).

HSP90 Abs activate the HSE-binding activity of HSF1 under nonstress conditions. (A) Left, gel mobility shift assay of uninjected oocytes (−) and HSP90 Ab-injected oocytes (MAb or PAb) that were incubated at nonshock temperatures (NS) or heat shocked for 1 h (HS). The positions of heat-activated or Ab-activated HSF1-HSE complexes (HSF1) and HSP90 Ab-supershifted complexes (HSF1 + ab) are indicated. Right, gel mobility shift assay showing the effects of coinjected bovine HSP90 on the formation of HSF1-HSE complexes. In the third lane (+HSP90), 50 ng of purified bovine HSP90 was injected into oocytes 2 h after injection of HSP90 PAbs, and extracts were made following incubation at a nonshock temperature for a further 1 h. ns, nonspecific binding. (B) Bottom, gel mobility shift assay of oocytes injected with PAbs against YY1 (Santa Cruz; see Materials and Methods). Top, comparison of the activation of HSF1 in uninjected (uninj) and sham-injected (H₂O) or HSF1 antiserum-injected (HSF1 PAb) () oocytes. (C) Recognition of HSF1 by microinjected HSP90 Abs occurred in vivo. A gel mobility shift assay was performed with uninjected (−) or HSP90 MAb-injected oocytes. In some lanes, HSP90 MAb or purified bovine HSP90 was added to extracts as indicated below the panel. HSP90 (1 μg) was added to Ab-injected oocytes at the time of homogenization (fifth lane from left), and HSP90 MAb or protein (1 μg) was added to uninjected heat-shocked oocyte extracts just prior to DNA-binding reactions (seventh and eighth lanes from left).