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A chemical cross-linking method for the analysis of binding partners of heat shock protein-90 in intact cells
 
Shaoming Song1, Sutapa Kole2, and Michel Bernier1
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Results and discussion

EGS-dependent formation of a stable 240-kDa protein complex encompassing Hsp90 (p240) in intact cells

The irreversible NH2 group-specific homobifunctional cross-linking reagent EGS was used initially to ascertain whether stable Hsp90-containing heteroprotein complexes could be generated in intact HepG2 cells. After cell lysis, cellular proteins were resolved by SDS-PAGE under reducing conditions and analyzed for Hsp90 by Western blot analysis using a monoclonal antibody that has specificity for both Hsp90α (HSP90AA1) and β (HSP90AB1) isoforms. A high molecular weight protein complex of ∼240 kDa encompassing Hsp90 (p240) was detected upon EGS cross-linking (Figure 1A). Neither the co-chaperones Hsp70 and p60Hop (Figure 1) nor p23 (data not shown) were found in p240 or in any other covalent multiprotein complexes. This heteroconjugate of Hsp90 was also detected in Hsp90 immunoprecipitates from EGS-treated HepG2 cells (Figure 1B). Of significance, exposure to EGS allowed the formation of p240 in 1321N1 astrocytoma cells and human PBMCs (see Figure 2C). Finally, the potency of EGS (16.1 Å) was compared with that of DMP (9.2 Å) and DSS (11.4 Å), two irreversible NH2 group-specific homobifunctional cross-linking reagents that have shorter spacer length. The results in Figure 1C showed that the distances between Hsp90 and interacting molecule(s) to generate p240 in situ requires a cross-linker with a 14 Å spacer.





A previous study by Rexin et al. (22) using the chemical cross-linkers DSS and EGS, demonstrated the formation of a constitutive hetero-oligomer complex of ≥300 kDa between Hsp90 and the nonactivated glucocorticoid receptor both in intact cells and in cell extracts. Similar analysis using DMP cross-linking showed the covalent association of a 59-kDa protein (p59) with Hsp90-glucocorticoid receptor complexes in cytosolic extracts (23). Other evidences showed that DSP and EGS cross-linking in cytosolic extracts covalently linked p59 to the Hsp90-glucocorticoid receptor complexes (24). In the latter study, Hsp90 was detected in a 2:1 ratio with respect to p59, which was later determined to be Hsp56 (FKBP4), an immunophilin of the FK506/rapamycin binding class (25). As such, Hsp56 associates with Hsp90 in untransformed mammalian steroid receptor complexes (25) and plays an important role in basic cellular processes involving protein folding and trafficking (26).

Intracellular heat shock proteins are among the most highly expressed proteins in normal and cancerous cells, and it is likely that the Hsp90-associated partner(s) is indeed a heat shock protein. Consistent with this notion is the fact that protein-protein interactions of Hsp90 and its co-chaperones are refractory to either the pharmacological inhibition of Hsp90 ATPase function or expression of Hsp90 mutants lacking the C-terminal homodimerization domain (27,28). In contrast, pharmacological inhibition of the intrinsic ATPase activity of Hsp90 has been found to disrupt client protein interaction with Hsp90 (2). Here, we present unequivocal evidence for p240 formation existing in intact cells treated with two structurally unrelated classes of Hsp90 inhibitors. The EGS-mediated accumulation of p240 was comparable in the absence or presence of 17-AAG and novobiocin in HepG2 and 1321N1 cells and in freshly isolated PBMCs (Figure 2, A–C). To independently assess whether the N-terminal ATP binding domain of Hsp90 has any role in orchestrating the formation of p240, HepG2 cells were transfected with two distinct epitope-tagged Hsp90α constructs that lack 94 amino acids at the N terminus (ΔNT).

These constructs, termed FLAG-ΔNT and Myc-ΔNT, express truncated recombinant forms of Hsp90α and, therefore, should give rise to a faster migrating complex when compared with the endogenous Hsp90α protein. Indeed, when anti-Myc immunoprecipitates of EGS-treated cells were subjected to Western blot analysis using the FLAG antibody, an ∼210-kDa multiprotein complex encompassing FLAG-ΔNT and Myc-ΔNT was observed (Figure 2D, lane 4). In the absence of EGS, there was co-sedimentation of FLAG-ΔNT (∼80 kDa) with Myc-ΔNT (Figure 2D, lane 3), consistent with the well-known noncovalent dimeric interaction of Hsp90 (1,2). Reciprocal immunoprecipitation was performed and gave similar results (data not shown), indicating that both the constitutive and EGS-mediated formation of Hsp90 dimers did not require the ATPase activity or binding motifs present in the N-terminal domain of Hsp90α. Furthermore, evidence of a heteromeric complex of cross-linked ΔNT-Hsp90α suggests that the ATPase function of Hsp90 is not necessary for the formation and stabilization of the quaternary structure of p240. Taken together, these results demonstrate that the EGS-mediated cross-linking event is specific and may provide an approach for the study of Hsp90-controlled processes that may occur in intact cells under physiological and stress-inducible conditions.

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