Biomedical subjects

L F Stancato

Publications and source records attributed to L F Stancato.

25 records · Page 2Linked to original sources

PubMed1994

The native v-Raf.hsp90.p50 heterocomplex contains a novel immunophilin of the FK506 binding class.

We have recently reported that v-Raf forms a native heterocomplex with rat heat shock protein (hsp) 90 and a 50-kDa phosphoprotein (p50) in stably transfected 3Y1 fibroblasts (Stancato, L. F., Chow, Y-H., Hutchison, K. A., Perdew, G. H., Jove, R., and Pratt, W. B. (1993) J. Biol. Chem. 268, 21711-21716). Several members of the nuclear receptor family exist in heterocomplexes containing hsp90 and various members of the immunophilin protein family, including hsp56, an immunophilin of the FK506 binding class (Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458). In this work, we have asked if Raf is also associated with an immunophilin. We have immunoadsorbed v-Raf from stably transfected rat 3Y1 fibroblasts and show that the immunoadsorbed v-Raf.hsp90.p50 heterocomplex binds the immunosuppressant drug [3H]FK506. The binding is of high affinity (KD 82 nM) and specific in the sense that it is competed by nonradioactive FK506 and rapamycin but not by cyclosporin A. The [3H]FK506 binding activity is eliminated when the heterocomplex proteins are dissociated from v-Raf. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for the immunophilin association. We have shown that hsp90 and p50 do not bind FK506, and the v-Raf heterocomplex does not contain any of the established FK506-binding proteins. Thus, we propose that Raf is associated with a novel immunophilin.

3T3 Cells↗

PubMed1993

Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.

Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

3T3 Cells↗

PubMed1993

Regulation of glucocorticoid receptor function through assembly of a receptor-heat shock protein complex.

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

Animals↗

PubMed1993

Differential effects of the reversible thiol-reactive agents arsenite and methyl methanethiosulfonate on steroid binding by the glucocorticoid receptor.

The hormone binding domain of the glucocorticoid receptor contains a unique vicinally spaced dithiol, and when it is bound by arsenite under conditions that are specific for reaction with vicinally spaced dithiols versus monothiols, steroid binding activity is eliminated [Simons, S. S., Jr., Chakraborti, P. K., & Cavanaugh, A. H. (1990) J. Biol. Chem. 265, 1938-1945]. The vicinally spaced dithiol lies in a region of the receptor that appears to be a contact site for hsp90, which is required for the high-affinity steroid binding conformation of the glucocorticoid receptor [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. As part of a long-term project to develop a vicinal dithiol-specific agent that will permit studies of ligand-induced conformational changes in this region of the receptor, we have examined here the differential effects of two reversible thiol-reactive agents, arsenite and MMTS. At low concentration, arsenite inactivates the steroid binding activity of the unliganded receptor in a vicinal dithiol-specific manner, whereas dissociation of steroid from untransformed, transformed, or DNA-bound transformed receptors occurs only at concentrations typical of monothiol interactions. MMTS produces a unique bimodal effect on the steroid binding capacity of the unliganded receptor at pH 9 that is pH-dependent and becomes essentially unimodal at physiological pH. Whereas arsenite disrupts the dexamethasone-receptor complex more readily than the triamcinolone acetonide-receptor complex, MMTS has the opposite effect. During treatment for 1 h at 0 degree C, neither reagent causes dissociation of hsp90 from the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The hsp56 immunophilin component of steroid receptor heterocomplexes: could this be the elusive nuclear localization signal-binding protein?

In many cells, the glucocorticoid receptor undergoes rapid steroid-mediated translocation from the cytoplasm to the nucleus, and this receptor is an excellent model for studying the mechanism of targeted protein movement through the cytoplasm. For such unidirectional movement to occur, the receptor must attach to a retrograde movement system in a manner that involves the nuclear localization signal. It is improbable that such attachment occurs via a direct protein-protein interaction between the receptor and the movement system; rather, one or more linker proteins are likely to be involved. As with other steroid receptors, the glucocorticoid receptor is associated with several other proteins in a heterocomplex. Two of these receptor-associated proteins are the heat shock proteins hsp90 and hsp56, and a third heat shock protein, hsp70, is required for assembly of the receptor heterocomplex. The hormone binding domain of the steroid receptors determines the interaction with both hsp90 and hsp70. Hsp56 is known to bind to hsp90, but its potential site, or sites, of interaction with the receptor are undefined. Hsp56 has recently been cloned and demonstrated to be an immunophilin of the FK506/rapamycin binding class. The immunophilins have peptidyl-prolyl isomerase activity but their cellular functions are unknown. Herein, we review the literature on the hsp56 immunophilin component of the receptor heterocomplex and present a rationale for hsp56 being the protein that determines the direction of receptor movement via a direct protein-protein interaction with the nuclear localization signal.

Animals↗

PubMed1992

The protein-protein complex between pp60v-src and hsp90 is stabilized by molybdate, vanadate, tungstate, and an endogenous cytosolic metal.

In a recent study demonstrating the cell-free reconstitution of the pp60v-src-hsp90 complex, we found that the tyrosine kinase-hsp90 complex is stabilized by molybdate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem. 267, 2902-2908). In this paper, we examine in detail the stabilization of this protein-protein interaction by transition metal oxyanions. The pp60v-src-hsp90 complex is stabilized by sodium molybdate with the same concentration dependence as the glucocorticoid receptor-hsp90 complex. As with the steroid receptor heterocomplexes, vanadate and tungstate also stabilize the pp60v-src-hsp90 interaction. Passage of cytosol through a Chelex-100 metal-chelating resin destabilizes the native pp60v-src-hsp90 complex, suggesting that the complex is normally stabilized by an endogenous metal factor. Readdition of either the heat-stable components of cytosol or a partially purified endogenous metal factor stabilizes the metal-depleted complex. Molybdate also stabilizes the presence of p50, a known hsp90-associated protein, in the pp60v-src heterocomplex. Given the identical effects of transition metal oxyanions on both pp60v-src- and steroid receptor-hsp90 complexes and the lack of any sequence homology between pp60v-src and the receptors, it seems very likely that it is the common component, hsp90, that contains the site of the metal interaction.

Animals↗

PubMed1991

Purification of the endogenous glucocorticoid receptor stabilizing factor.

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.

Animals↗