Modulation of Nuclear Factor kappa B by FK506-Binding Proteins

ERLEJMAN A, DE LEO A, MOLINARI A, MAZAIRA G, GALIGNIANA MD

Porto Alegre

Conferencia; IX Cell Stress Society International Meeting on the Molecular Biology of Stress Responses; 2012

CSSI

Introduction. NF-kB is a transcriptional regulator of basic cellular functions. Abnormal NF-kB activities have been implicated in various disease states such as cancer, inflammation, autoimmunity, diabetes, AIDS, neurodegeneration and stroke. Canonical NF-kB is a heterodimer of p65/RelA with p50 subunits that reside in the cytoplasm as inactive complexes bound to IêB. Several stimuli promote IkB phosphorylation followed by its degradation by the proteasome. IkB release permits the nuclear translocation of NF-B. In previous studies, we reported that the Hsp90-binding immunophilins FKBP51 and FKBP52 are responsible in a mutually exclusive fashion of the retrotransport mechanism of glucocorticoid receptors (GR) and the GR-dependent transcriptional activity. These FK506 binding proteins (FKBPs) have two key sequences, the TPR domain, through which they bind to Hsp90, and the PPIase domain, where FK506 and dynein bind. To date, the biological functions of both FKBPs are poorly understood and remain elusive. Objectives. We hypothesized that both FKBPs could regulate the relocalization and biological function of NF-kB, a transcription factor that shows similar trafficking requirements to the GR. Methodology. HEK-293T cells were transfected with FKBP51, FKBP52 and/or mutant constructs. Cells were stimulated with PMA and the NF-kB subcellular localization was visualized by IFI. Transcriptional activity was measured by a gene reporter-assay, and the interaction with promoter sites was evidenced by EMSA. The putative NF-kB/FKBP association was investigated by coimmunoprecipitation (co-IP) assays. Results. PMA-induced NF-kB activation was abrogated by overexpression of FKBP51, which was recruited by promoter sites. This inhibition was reversed by overexpression of the TPR domain. On the other hand, FKBP52 showed a notorious stimulating effect that was abolished by FK506 or a TPR domain mutant unable to interact with Hsp90. The latter treatments did not affect the FKBP51 inhibitory action. The relevance of the PPIase activity of FKBP52 was confirmed by the lack of effect of an inactive PPIase mutant. Competition assays with increasing amounts of FKBP52 reversed the inhibitory effect of FKBP51, suggesting as mutually exclusive properties as those observed with GR. Importantly, Rel A co-IP with FKBP51 in untreated cells, whereas PMA stimulation promoted its exchange with FKBP52. Interestingly, the nuclear translocation rate of Rel A was delayed by FKBP51, whereas Rel A nuclear retention was favored by FKBP52. Conclusions We postulate that FKBP51 is an inhibitory factor of NF-kB, an effect that is competed by FKBP52, a strong stimulator whose effect depends on its intrinsic PPIase activity. Taken together, these observations raise the possibility that NFkB function may be regulated according to the expression balance of both chaperones in a given cell type or condition.