Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Abstract

. 111: [807][808][809][810][811][812][813][814][815][816] 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30°C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.Large karyophilic proteins are imported into nuclei through the nuclear pore complex (NPC) in a process that can be divided into at least two steps (4,10,43,63). In the first step of the nuclear translocation process, proteins destined for import bind to the NPC via their association with soluble receptor proteins in an ATP-independent, N-ethylmaleimide (NEM)-sensitive reaction (44,45,55). The second step in this process is an ATP-dependent translocation through the pore. The translocation step requires both soluble factors (41) and components of the NPC (14, 66), which include a family of glycoproteins with 0-linked N-acetylglucosamine modifications (15,26).Karyophilic proteins generally contain nuclear localization signal sequences (NLSs) (30, 32), sequences of amino acids that serve as recognition sites for the soluble receptor proteins (2, 33, 72). In some cases, proteins destined for nuclear import which lack discernible NLSs can be cotransported into nuclei presumedly via stable interactions with NLS-containing proteins (22,65). Although the initial prototypic NLS identified within the simian virus 40 (SV40) large tumor antigen (TAg) is composed of a contiguous stretch of basic residues flanked by prolines (30, 32), most other NLSs appear to be more complex and bipartite in nature (11,56 61), and an unrelated 70-kDa phosphoprotein from S. cerevisiae (68). While the functions of the 54/56-kDa proteins in nuclear import are sensitive to NEM in vitro, it appears that they are not the only NEM-sensitive cytosolic components that ...

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus

“…The nuclear localization of these receptors was also confirmed based on the immunohistochemical observations of rat reproductive organs. Glucocorticoid receptor (GR) shows the ligand-dependent translocation into the nucleus from the cytosol (Yang & DeFranco 1994, Sackey et al 1996. In contrast, the localization of the immunoreactive products for both ER and ER in transfected 293T cells was confined to the nuclei regardless of the presence of E2, implying that the nuclear localization of these receptors is ligand independent (Welshons et al 1984).…”

Neuronal Hypoxia Induces Hsp40-Mediated Nuclear Import of Type 3 Deiodinase As an Adaptive Mechanism to Reduce Cellular Metabolism

“…6 B) that regulates complex formation between Hsc70 and client proteins by binding and delivering specific proteins to Hsc70 or by stabilizing Hsc70-polypeptide complexes (Fan et al, 2003;Qiu et al, 2006). Hsp40-directed client proteins to Hsc70 is known to mediate the nuclear import of glucocorticoid receptors (Yang and DeFranco, 1994), HIV-1 preintegration complex (Agostini et al, 2000) and karyopherin ␣ (Agostini et al, 2000). Moreover, the Hsp40 family member DNAJB6 is also involved in nuclear import of HIV-2 preintegration complex (Cheng et al, 2008) and localization of T-Cell quiescence protein, Schlafen-1 in the nucleus (Zhang et al, 2008).…”

The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone

“…Several workers have reported Hsp70 association with T to be dependent upon the presence of the T N-terminal segment and that the sequences necessary for this interaction have been delimited to residues 1-97 including the J-domain (58,59). In addition, SV40 T nuclear localization appears to be critically dependent upon cytoplasmic Hsp70 or Hsc70 (60)(61)(62).…”