Hsp90 is involved in the formation of P-bodies and stress granules

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Abstract

Previously, we found that treatment of cells with the Hsp90 inhibitor geldanamycin (GA) leads to a substantial reduction in the number of processing bodies (P-bodies), and also alters the size and subcellular localization of stress granules. These findings imply that the chaperone activity of Hsp90 is involved in the formation of P-bodies and stress granules. To verify these observations, we examined whether another Hsp90 inhibitor radicicol (RA) affected P-bodies and stress granules. Treatment with RA reduced the level of the Hsp90 client protein Argonaute 2 and the number of P-bodies. Although stress granules still assembled in RA-treated cells upon heat shock, they were smaller and more dispersed in the cytoplasm than those in untreated cells. Furthermore eIF4E and eIF4E-transporter were dissociated selectively from stress granules in RA-treated cells. These observations were comparable to those obtained upon treatment with GA in our previous work. Thus, we conclude that abrogation of the chaperone activity of Hsp90 affects P-body formation and the integrity of stress granules.

Highlights

► Treatment with RA reduces the amount of Ago2 protein and the number of P-bodies. ► The size and localization of stress granules are altered in RA-treated cells. ► eIF4E and 4E-T are dissociated selectively from stress granules in RA-treated cells. ► Hsp90 is required for the formation of P-bodies and stress granules.

Introduction

The heat shock protein Hsp90 is one of the most conserved molecular chaperones and is essential for living cells [1], [2], [3], [4], [5], [6], [7], [8]. Hsp90 facilitates the folding, assembly and stabilization of a wide range of client proteins including protein kinases and transcription factors, and the number of listed Hsp90 clients is continuously increasing [1], [2], [3], [4], [5], [6], [7], [8]. Geldanamycin (GA) is an inhibitor of Hsp90. Recently, we [9] and other groups [10], [11] have shown that GA treatment causes the disappearance of processing bodies (P-bodies), cytoplasmic granules in which translationally inactive mRNAs are thought to colocalize with the machinery for translational repression and mRNA decay [12], [13], [14], [15], [16]. In addition, we found that inhibition of Hsp90 with GA also alters the size and subcellular localization of stress granules, which are another class of RNA granule [12], [17], [18]. Treatment with GA results in the specific loss of the mRNA cap-binding protein eIF4E and its binding partner eIF4E-transporter (4E-T) from these granules; in addition, the interaction between eIF4E and the translation initiation factor eIF4G is reduced [9]. These findings imply that Hsp90 is involved in the process of translation initiation. Moreover, it has now become evident that Hsp90 mediates the ATP-dependent formation of the RNA-induced silencing complex by facilitating the loading of small RNA duplexes onto Argonaute (Ago) proteins [19], [20], [21]. Thus, Hsp90 chaperone activity contributes to the posttranscriptional regulation of mRNAs.

Hsp90 exhibits molecular chaperone activity by conformational cycling between loading and release of client proteins in an ATPase-coupled manner [1], [2], [3], [4], [5], [6], [7], [8]. The ATP-driven chaperone cycle of Hsp90 requires the coordinated assistance of a range of co-chaperone proteins, which assemble into an activated multichaperone complex [1], [2], [3], [4], [5], [6], [7], [8]. This multichaperone complex is more susceptible to inhibitors, such as GA and radicicol (RA), than the uncomplexed form of Hsp90 [22]. The determination of co-crystal structures has revealed that GA and RA act as nucleotide mimetics and bind to the deep nucleotide pocket of the N-terminal domain of Hsp90, thereby blocking the Hsp90 chaperone cycle [5], [8], [23], [24].

It has been shown that the treatment of cells with GA leads to the formation of superoxide radicals, which in turn result in cell toxicity through a mechanism that is completely independent of Hsp90 inhibition. Thus, GA is not necessarily a specific inhibitor of Hsp90 [25]. Another compound RA, which is structurally distinct from GA [8], [23], [24], also binds Hsp90, although it does bind ATP citrate lyase [26] and DNA topoisomerase VI [27] as well. Therefore, to verify whether the effects of GA on the formation of P-bodies and stress granules [9] are attributable to abrogation of the chaperone activity of Hsp90, we examined whether our previous observations that were obtained using GA could be reproduced by treating cells with RA.

Section snippets

Cell culture and immunocytochemistry

HeLa S3 cells were maintained as described previously [28]. Cells that had been grown on coverslips in 24-well plates were treated with 2 μM GA (Sigma), 1, 2 or 4 μM RA (Sigma) or dimethylsulfoxide for 24 h and fixed for immunocytochemistry. For heat stress, the 24-well plates were floated in a water bath in a CO2 incubator at 44 °C for 30 min just before fixation [29]. Immunocytochemistry was performed as described previously [30], [31].

Antibodies

The rabbit polyclonal antibodies against Dcp1a [30] and YB-1

The number of P-bodies is decreased by treatment with RA

By inhibiting Hsp90, RA, like GA [33], [34], destabilizes Hsp90 client proteins and causes the degradation of these proteins [35]. To establish appropriate conditions for the RA treatment, the concentration of RA was varied and the effect on the amount of Ago2 protein, which is an Hsp90 client protein, was examined. At a concentration of 1 μM, RA did not seem to affect the level of Ago2 protein in HeLa cells (Fig. 1, 1 μM RA). However, 2 μM RA induced a significant decrease in Ago2 and this

Acknowledgments

We would like to thank Dr. Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science) for helpful discussion. This work was supported in part by a Grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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