Hsp90-binding immunophilins link p53 to dynein during p53 transport to the nucleus

The tumor suppressor protein p53 is known to be transported to the nucleus along microtubular tracks by cytoplasmic dynein. However, the connection between p53 and the dynein motor protein complex has not been established. Here, we show that hsp90·binding immunophilins link p53·hsp90 complexes to dynein and that prevention of that linkage in vivo inhibits the nuclear movement of p53. First, we show that p53·hsp90 heterocomplexes from DLD-1 human colon cancer cells contain an immunophilin (FKBP52, CyP-40, or PP5) as well as dynein. p53·hsp90·immunophilin·dynein complexes can be formed by incubating immunopurified p53 with rabbit reticulocyte lysate, and we show by peptide competition that the immunophilins link via their tetratricopeptide repeat domains to p53-bound hsp90 and by means of their PPIase domains to the dynein complex. The linkage of immunophilins to the dynein motor is indirect by means of the dynamitin component of the dynein-associated dynactin complex, and we show that purified FKBP52 binds directly by means of its PPIase domain to purified dynamitin. By using a temperature-sensitive mutant of p53 where cytoplasmic-nuclear movement occurs by shift to permissive temperature, we show that p53 movement is impeded when p53 binding to hsp90 is inhibited by the hsp90 inhibitor radicicol. Also, nuclear movement of p53 is inhibited when immunophilin binding to dynein is competed for by expression of a PPIase domain fragment in the same manner as when dynein linkage to cargo is dissociated by expression of dynamitin. This is the first demonstration of the linkage between an hsp90-chaperoned transcription factor and the system for its retrograde movement to the nucleus both in vitro and in vivo

Impairment of mineralocorticoid receptor (MR)-dependent biological response by oxidative stress and aging: Correlation with post-translational modification of MR and decreased ADP-ribosylatable level of elongation factor 2 in kidney cells

Acute and chronic treatments of mice with the glutathione-depleting agent, L-buthionine-(SR)-sulfoximine (BSO), impaired the mineralocorticoid receptor (MR)-dependent biological response by inhibiting aldosterone binding. This steroid-binding inhibition was fully reversed when reducing agents were added to kidney cytosol obtained from mice treated for 5 h, but it was only partially reversed in cytosol obtained from mice treated for 10 days. Although the oligomeric structure of the MR-hsp90 heterocomplex was always unaffected, a decreased amount of MR protein was evidenced after the long term treatment. Such a deleterious effect was correlated with a post-translational modification of MR, as demonstrated by an increased level of receptor carbonylation. In addition, a failure at the elongation/termination step was also observed during the receptor translation process in a reticulocyte lysate system. Thus, a high polyribosomes/monomers ratio and both increased proteolysis and decreased ADP-ribosylatable concentration of elongation factor 2 (EF-2) were shown. Importantly, similar observations were also performed in vivo after depletion of glutathione. Notwithstanding the EF-2 functional disruption, not all renal proteins were equally affected as the MR. Interestingly, both EF-2 and MR expressed in old mice were similarly affected as in L-buthionine-(SR)-sulfoximine-treated young mice. We therefore propose that a dramatic depletion of glutathione in kidney cells mimics the cumulative effect of aging which, at the end, may lead to a renal mineralocorticoid dysfunction

The hsp90-FKBP52 complex links the mineralocorticoid receptor to motor proteins and persists bound to the receptor in early nuclear events

In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm. Copyright © 2010, American Society for Microbiology. All Rights Reserved.Fil:Monte, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Modification of an essential amino group in the mineralocorticoid receptor evidences a differential conformational change of the receptor protein upon binding of antagonists, natural agonists and the synthetic agonist 11,19-oxidoprogesterone

The alkylation of amino groups of the mineralocorticoid receptor (MR) with pyridoxal 5′-phosphate or 2,4,6-trinitrobenzenesulphonate (TNBS) under controlled conditions modifies only one lysyl residue, which accounts for a 70% inhibition of steroid binding capacity. The Kd of aldosterone for MR is not affected by the treatment, but the total number of binding sites is greatly decreased. The modified receptor is capable of dynamically conserving its association with the hsp90-based heterocomplex. Importantly, the binding of natural agonists protects the hormone binding capacity of the MR from the inactivating action of alkylating agents. In contrast, antagonistic steroids are totally incapable of providing such protection. Like the antagonistic ligands, and despite its potent mineralocorticoid biological effect, the sole MR specific synthetic agonist known to date, 11,19-oxidoprogesterone (11-OP), shows no protective effect upon treatment of the MR with pyridoxal 5′-phosphate or TNBS. Limited digestion of the MR with α-chymotrypsin generates a 34 kDa fragment, which becomes totally resistant to digestion upon binding of natural agonists, but not upon binding of antagonists. Interestingly, the synthetic 21-deoxypregnanesteroid 11-OP exhibits an intermediate pattern of proteolytic degradation, suggesting that the conformational change generated in the MR is not equivalent to that induced by antagonists or natural agonists. We conclude that in the first steps of activation, the MR changes its conformation upon binding of the ligand. However, the nature of this conformational change depends on the nature of the ligand. The experimental evidence shown in this work suggests that a single lysyl group can determine the hormone specificity of the MR. © 2002 Elsevier Science B.V. All rights reserved.Fil:Ghini, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Burton, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina