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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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Subcellular distribution of p240 in EGS-treated cells

In addition to its cytosolic expression, Hsp90 is detected in mitochondria and on the surface of normal and tumor cell membranes (8,10-12)(29). Here, mitochondrial and cytosolic fractions from control and EGS-treated HepG2 cells were prepared and analyzed by Western blot analysis for the presence of p240 (Figure 3A). Both subcellular compartments contained monomeric Hsp90 and p240. The blots were reprobed with the mitochondrial marker, complex V, and the cytosolic marker, IκBα, to confirm the quality of our cell fractionation. Furthermore, when crude membranes (100,000× g pellets) from EGS-treated cells were immunoblotted with anti-Hsp90 antibody, there was clear expression of p240, although to levels that were significantly lower when compared with the cytosolic fraction (Figure 3B). As expected, the crude membrane fraction stained positively for EGF receptors (Figure 3B, lower panel).



To assess whether p240 could accumulate at the cell surface facing the extracellular milieu, cells were treated with the cell-impermeant EGS analog, sulfo-EGS (Figure 3C). The results indicate the detection of p240 in sulfo-EGS–treated cells which, when normalized to the amount of monomeric Hsp90, yielded a 10-fold lower abundance as compared with cells treated with EGS (Figure 3C, upper panel). To independently verify the presence of a subpopulation of p240 on the cell surface, we compared the effect of sulfo-EGS on the formation of p240 and HSF1 trimers in HepG2 cells. HSF1 is a cytosolic transcription factor that requires trimerization for its nuclear translocation and DNA binding properties. Consistent with our recent observation (30), constitutive formation of HSF1 trimers was observed in EGS-treated cells; in contrast, sulfo-EGS was unable to promote HSF1 oligomerization (Figure 3C, lower panel, lane 3 versus 2) while inducing the accumulation of p240.

Recent studies have demonstrated that Hsp90 gets exported at the cell surface and is secreted in conditioned media by both normal and tumor cells (reviewed in Reference 31). The reported targets for extracellular Hsp90α include the co-chaperones Hsp70, p60Hop, p23, and others in breast cancer cells; this co-chaperone complex increases matrix metalloproteinase 2 (MMP-2) binding to Hsp90α and assists in MMP-2 activation, thereby promoting cancer cell migration and invasion in vitro (32). The detection of p240 at the cell surface of EGS-treated HepG2 cells may have important biological implications, as extracellular Hsp90 has been reported to dock with membrane-bound CD91/LRP1, hyaluronan receptor CD44, and tyrosine kinase receptors (31,33), as well as acting as negative regulator for the activation of latent TGF-β1 (34).

Role of FLNa in the formation and subcellular distribution of p240

Recent proteomic approaches have identified FLNa (also known as ABP280) as a novel interaction partner of Hsp90 (16). FLNa, a member of the nonmuscle actin binding protein family, is a widely expressed molecular scaffold protein that regulates signaling events such as subcellular localization of signaling proteins through changes in cytoskeletal actin dynamics (35). To ascertain the role of FLNa in the formation and subcellular distribution of p240, siRNA-mediated knockdown of FLNa was carried out in HepG2 cells, as evidenced by the potent and selective suppression of FLNa protein levels (Figure 4A). Under these conditions, there was no significant difference in the immunodetection (Figure 4B) and cell surface localization (Figure 4C) of p240 when cells that were transfected with either FLNa siRNA or nonsilencing control siRNA were treated with sulfo-EGS. Moreover, human M2 melanoma cells that lack FLNa also exhibited EGS-dependent accumulation of p240 (data not shown). Therefore, FLNa does not appear to be a key mediator of the recruitment, subcellular compartmentalization and/or dynamic trafficking of p240 in EGS-treated cells.



To conclude, the demonstration that p240 is stoichiometrically abundant is of significance and indicates that the interaction partner(s) is also abundantly expressed. Because of the lack of MS/proteomics data, we cannot rule out the possibility that p240 may consist of an Hsp90 dimer with aberrant electrophoretic mobility along with the existence of populations of heteromers. However, given the fact that the electrophoretic mobility of p240 is significantly slower than that of the DMS-linked Hsp90 dimers (36), it is likely that a distinct component of ∼60 kDa is present in p240 in association with two Hsp90 molecules. Moreover, our observation that only EGS with its 14-Å spacer was able to stabilize covalently p240 in intact cells is in contrast with what was observed by others using cytosolic extracts and chemical cross-linkers with shorter spacer arms (22-25). The simplest interpretation for these results is that the quaternary structure of the cytosolic Hsp90 hetero-oligomeric complexes and/or availability of reactive amino groups (e.g., appropriate average molecular spacing and geometry) may differ as a consequence of cell homogeneization. Nevertheless, the specificity of the cross-linking event observed here may provide an approach to expand the range of tools available to the study of the Hsp90 interactome.

Acknowledgments

We thank the National Institute on Aging (NIA), National Institutes of Health (NIH) Apheresis Unit, and the clinical core laboratory for providing human blood from normal donors. This research was supported entirely by the Intramural Research Program of the NIH, NIA. This paper is subject to the NIH Public Access Policy.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Michel Bernier, Biomedical Research Center, Laboratory of Clinical Investigation, National Institute on Aging, NIH, 251 Bayview Boulevard, Suite 100, Baltimore, MD, USA. e-mail address: [email protected]">[email protected]

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