The steroid hormones are used extensively to treat cancers of the breast, endometrium, prostate, and lymphoid tissues. These hormones act by binding to receptors that must become precisely located at sites within the nucleus where they regulate transcription from a limited number of genes. In the hormone-free cell, the glucocorticoid receptor (GR) and the sex hormone receptors exist in a heteroprotein complex that contains three heat shock proteins-hsp90, hsp70 and hsp56. These three hsps are chaperone proteins that form a complex with each other independent of the presence of receptors and we postulate that the complex is involved in the very essential cellular functions of protein folding and trafficking. From our studies using the GR as a model system, we propose that hsp90 and hsp56 are critical factors involved in receptor transport to (and perhaps subsequently within) the nucleus; the notion being that heat shock protein complex may act as a transport particle or transportosome to which steroid receptors and a wide variety of proteins are bound as they are transported along cytoskeleton-based movement systems within the cell. Because the steroid receptors must move in some ordered fashion under the direction of their nuclear localization signals (NLS) to reach the precise nuclear loci where transcriptional regulation occurs, the fundamental biology of the linked phenomena of receptor folding and trafficking must be defined. The first set of specific aims in this proposal focuses on mechanistic aspects of the GR translocation model. The heat shock protein complex was identified by us during the current granting period when we showed that immunoadsorption of hsp56 from human lymphoblast cytosol results in coimmunoabsorption of hsp70 and hsp90. Hsp56 was recently shown to be an immunophilin that binds FK506.
Specific aim l uses protein cross-linking and peptide competition approaches to define the site of hsp56 binding to the GR. I present a detailed model in support of the notion that the NLS of the GR (NL1) may be that site of interaction.
Specific aim 2 pursues our preliminary observation that hsp56 is the component of the GR heterocomplex that interacts with dynein, the motor component of the microtubule-based trafficking system supporting movement toward the nucleus. We have found that the hsp70 component of the hsp complex does not bind to hsp90 without the help of a factor in reticulocyte lysate and specific aim 3 focuses on identifying this factor. We have shown that the reticulocyte lysate system that forms the GR-hsp heterocomplex also dissociates the transformed GR from DNA, returning the receptor to the untransformed, hsp90-bound, non-DNA-binding state.
Specific aim 4 focuses on determining the mechanism of this GR """"""""unwrapping"""""""" process and whether it applies to GR bound to DNA after steroid addition to intact cells. The second set of specific aims asks whether the transportosome model can be expanded to other proteins that undergo trafficking to the nucleus, such as heat shock factor studied in specific aim 5. To determine if regulation of other proteins besides nuclear receptors occurs through interaction with hsp90, we ask in specific aim 6 if EGF affects the binding of Raf kinase to hsp90 and in specific aim 7 whether protein kinase C binds to hsp90 in a manner that is modified by phorbol ester.
Showing the most recent 10 out of 83 publications
