The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Abstract

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.Chaperones are essential for cell life because of their involvement in the folding of newly synthesized proteins. Among the chaperones, the heat-shock protein of 90 kDa (Hsp90) 3 is one of the most abundant and ubiquitously expressed cellular proteins (10 -50 M (1)) with a main function of helping other proteins maintain their conformation in an active state. Several client proteins of Hsp90 involved in cell proliferation and apoptosis (such as protein kinases and transcription factors) have been described (2-5). Although Hsp90 is fundamental for the life of normal cells, it also protects mutated proteins against cellular degradation, thus promoting cancer cell survival (6). Therefore, considerable efforts have been made to find inhibitors of its activity (7,8).Over the past decade several studies have suggested a possible relationship between Hsp90 and the microtubule cytoskeleton (9 -15). Tubulin is a highly concentrated cellular protein (10 -20 M (16)) and is the building block of microtubules, which are involved in several crucial processes such as cell division and morphogenesis, compartmentalization, and organelle movement. Its fundamental role in the architecture and functioning of the mitotic spindle has also made this protein a classical target of antimitotic drugs. A direct interaction between Hsp90 and tubulin has previously been described (9), but Hsp90 is also known to interact with other chaperones and co-chaperones to perform its function (17-19). Therefore, whether Hsp90 can protect tubulin against denaturation in the absence of other factors remains unknown.In this study we investigated the direct interaction between tubulin and Hsp90, both purified from porcine brain. Tubulin was subjected to thermal denaturation in the absence and in the pres...

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