Serine / threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling

David A Dean,Ileana V Aragon,Beth Miller,Sandro Rusconi,Nicholas M Dean,Richard E Honkanen,Mark Swingle,Gudrun Urban,Manuel Bueno

doi:10.1186/1471-2121-2-6

Abstract

BackgroundIn most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.ResultsTo further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP.ConclusionThese studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

Highlights

In most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm
Whereas assays conducted with phosphatases type 1 (PP1) and PP2A were conducted with a concentration of enzyme that is diluted below the titration endpoint because the activity of phosphatase type 5 (PP5) upon further dilution was below that necessary for accurate quantification, we could not establish a clear titration endpoint with PP5
Nuclear accumulation of GR-green fluorescent fusion protein (GR-GFP) was observed when the expression of PP5 was suppressed by treatment with ISIS 15534 after ~ 24 hours

Summary

Introduction

In most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GRcomplex regulates the transcription of GR-responsive genes. Little is known about the molecular machinery that regulates steroid receptor movement through the cytoplasm and into the nucleus, several studies suggest that movement is influenced by reversible phosphorylation. Evidence for this originated from the studies of Qi et al [5, 6], which revealed that the hormone insensitivity produced by cellular transformation with v-mos (a serine/threonine protein kinase that acts as an oncogene) results from both a decrease in the nuclear retention of liganded receptor and a decrease in the reutilization of GR protein that cycles back into the cytoplasm. Recent studies with okadaic acid suggest phosphorylation alters the high affinity binding of GR to hsp-90, and that an intact cytoskeleton is required for ligand-activated GR translocation through the cytoplasm to the nucleus [8]

Methods

Results

Discussion

Conclusion