Protein 14-3-3σ Interacts with and Favors Cytoplasmic Subcellular Localization of the Glucocorticoid Receptor, Acting as a Negative Regulator of the Glucocorticoid Signaling Pathway

Abstract

The glucocorticoid receptor (GR) ␣ interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GR␣ in a cell line replete with or devoid of 14-3-3. We found that endogenous 14-3-3 helped localize green fluorescent proteinfused GR␣ in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3 also suppressed the transcriptional activity of GR␣ on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3 inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GR␣, whereas introduction of a mutation inactivating the binding activity of 14-3-3 to some of its partner proteins did not. 14-3-3 bound the ligand-binding domain of GR␣ through its COOH-terminal portion, in a partially liganddependent fashion, while it did not interact with "ligand-binding domain" of GR␤ at all. These results suggest that 14-3-3 functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GR␣. Conversely, through its 14-3-3 protein interactions, GR␣ may influence these processes. The glucocorticoid receptor (GR)1 belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins and mediates the diverse and pivotal actions of glucocorticoids in the maintenance of resting and stress homeostasis (1). The human GR consists of two highly homologous isoforms, ␣ and ␤, produced by alternative use of exon 9 ␣ or ␤ of the GR gene. GR␣ represents the classic glucocorticoid receptor, which binds glucocorticoids and mediates almost all known glucocorticoid effects, while GR␤ does not bind ligand, has dominant negative activity upon GR␣ and unclear physiologic role(s) (2). GR␣, in the ligand-free condition, resides primarily in the cytoplasm in the form of a hetero-oligomer with several heat shock proteins (hsps) and related molecules (2). Upon hormone binding, GR␣ undergoes a conformational change, dissociates from the hsps and translocates into the nucleus depending on nuclear translocation signals 1 and 2 (3-5). Nuclear translocation signal 1 catalyzes rapid transport of the GR through the nuclear pore, employing the importin-mediated pathway, while nuclear translocation signal 2 contributes to a slower traffic via as yet unknown mechanisms (6).After entering the nucleus, GR␣ binds...

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Pratt and colleagues have demonstrated that components of the apoGR complex bind cytoskeletal factors (Galigniana et al, 2001). Alternatively, Kino et al (2003) have suggested that binding to the 14-3-3 protein helps tether apoGR to the cytoplasmic compartment. Given these observations, it would be interesting to examine the intracellular distribution of GR in the absence of hormone by electron microscopy.…”

Rho GDP dissociation inhibitor α interacts with estrogen receptor α and influences estrogen responsiveness

“…It seems plausible that cdc42 isoform1, a RhoGTPase family member with a polybasic region in its C-terminal end that can function as a nuclear localization signal (Lanning et al 2003, Williams 2003, might perform this function since we identified both isoforms 1 and 2 of cdc42 in our agarose gel purification experiments as ERa-associated proteins (Schultz-Norton et al 2007). Alternatively, RhoGDIa could be accompanied by a protein such as 14-3-3, which binds and helps to redistribute an array of signaling proteins (Fu et al 2000, Kino et al 2003, Diviani et al 2004. It should also be noted that the 28 kDa RhoGDIa is theoretically small enough to traverse the nuclear pores unaccompanied.…”

Proteomic identification of 14-3-3 sigma as a common component of the androgen receptor and the epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12

“…The GFP-fused 14-3-3s plasmid (Kino et al, 2003) was kindly provided by Dr T Kino (NIH, Bethesda, MD, USA). Transient transfection was performed using lipofectin (Life Technologies, Rockville, MD, USA), according to the manufacturer's protocol.…”