Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein

Jennifer M Harrell,Patrick J.M Murphy,Yoshihiro Morishima,Haifeng Chen,John F Mansfield,Mario D Galigniana,William B Pratt

doi:10.1074/jbc.m406863200

Abstract

Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR.hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR.hsp90.immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR(-) L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.

Highlights

As the initial step in their action, transcription factors, such as steroid receptors, p53, and HSF1, must move in a targeted manner through the cytoplasm to the nucleus
GR1⁄7hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus
Inhibition of glucocorticoid receptors (GR) Transport by Expression of Dynamitin—To determine whether cytoplasmic dynein is required for rapid translocation to the nucleus, dexamethasone-dependent movement of the endogenous GR was examined in 3T3 mouse fibroblasts expressing myc-dynamitin

Summary

Introduction

As the initial step in their action, transcription factors, such as steroid receptors, p53, and HSF1, must move in a targeted manner through the cytoplasm to the nucleus. Because the glucocorticoid receptor (GR) moves rapidly and quantitatively from the cytoplasm to the nucleus in a ligand-dependent manner, it has been a useful model for studying the movement process The GR forms heterocomplexes with hsp, and experiments testing the effects of molybdate and geldanamycin on GR trafficking have led to the concept that a dynamic process of receptor1⁄7hsp complex assembly-disassembly is required for rapid nuclear translocation [3, 4]. GR1⁄7hsp heterocomplexes contain one of three TPR domain immunophilins (FKBP52, FKBP51, and cyclophilin 40) or protein phosphatase 5 (PP5), a protein phosphatase that contains TPR and PPIase homology domains (reviewed in Ref. 1). It has not been demonstrated that receptor movement is dynein-dependent

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