The steroid hormones are used extensively to treat cancers of 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. It is not known how signaling proteins, such as steroid receptors, STAT proteins or MAP kinases, move in precisely targeted manner through the cytoplasm to their nuclear sites of action, and this constitutes a fundamental gap in the modeling of signaling pathways for a wide variety of hormones, growth factors, and drugs. Because movement of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus is initiated by addition of steroid, the GR is a very useful model for studying targeted movement of a signaling protein through the cytoplasm. Over the past several years of work on this grant, we have made a number of observations that support a model of GR movement in which the steroid-transformed receptor interacts dynamically with a machinery that facilitates receptor movement through the cytoplasm along cytoskeletal tracts to the nucleus. An okadaic acid-sensitive phosphatase activity is required for receptor transformation, and rapid movement of the transformed receptor requires its dynamic interaction with the hsp90 and immunophilin components of the comparatively """"""""stable"""""""" 9S untransformed receptor heterocomplex. In this funding period, we wish to determine if the serine/threonine phosphatase PP5, which binds directly to hsp90 and is a component of untransformed GR.hsp90 heterocomplexes, is the okadaic acid-sensitive phosphatase required for GR movement and if PP5 dephosphorylates the GR in an hsp90-dependent manner. The FK506 binding protein FKBP52 is the major immunophilin in GR.hsp90 heterocomplexes, and we want to determine if the peptidylprolyl isomerase domain of FKBP52 is involved in rapid GR movement to the nucleus. FKBP52 binds directly via its tetratricopeptide repeat domain to hsp90, it binds directly to the GR, and its immunoadosorption is accompanied by coimmunoadsorption of cytosolic dynein. We now want to determine what regions of FKBP52 interact with the GR and with the microtubule-associated motor protein dynein. Another major focus is to establish a permeabilized cell system, in which we can reconstitute energy-dependent and cytosol-dependent GR transfer to the nucleus to permit direct study of the components of a movement system.
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