HMGN2 Inducibly Binds a Novel Transactivation Domain in Nuclear PRLr to Coordinate Stat5a-Mediated Transcription

Abstract

The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

“…41 Each cell line was plated at 70% confluency in six-well plates and 24 hours post-plating was serum starved for an additional 24 hours. Triplicate samples were either treated with 250 ng/mL PRL for 2 hours or left untreated.…”

“…41 Knockdown cell lines were generated using a predesigned HMGN2 short hairpin RNA (shRNA) pRFP retroviral vector (Origene, Rockville, MD); PRLr shRNA was designed and cloned into a pRFP vector [sequences: HMGN2, 5 0 -GTGTCAGGCAATCTGGACT-TTCCAGTGAT-3 0 ; short hairpin PRLr (shPRLr), 5 0 -CAA-CTGCATAACCTTTACACT-3 0 ]. Recombinant retroviruses for pBabe, PRLr, PRLrYDmut, Scrambled shRNA, and shPRLr were generated using transfection of retroviral vectors with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 293T/ 17 cells, along with pVSVG and pGalPol vectors.…”

“…Given our previous data that expression of a transactivationdeficient PRLr impaired PRL-induced CISH expression, 41 we hypothesized that PRLrYDmut would impair global PRL-induced gene expression. To test this hypothesis, we used MCF7-PRLr, MCF7-PRLrYDmut, or MCF7-empty vector stable transfectants (herein referred to as PRLr, PRLrYDmut, and empty, respectively), as described in our previous report.…”

“…Expression of a transactivation-deficient mutant (Y406F/D411A; herein referred to as PRLrYDmut) significantly diminished both Gal4 reporter transactivation and Stat5a-mediated gene expression. 41 Preliminary evidence also suggested that the transactivation properties of the PRLr correlate to a tumorigenic phenotype in vitro, as assessed by anchorage-independent growth.…”

“…41 In addition, we identified a novel transactivation domain (TAD) within the PRLr and found that residues Y406 and D411 were necessary for PRLr-mediated transactivation. Expression of a transactivation-deficient mutant (Y406F/D411A; herein referred to as PRLrYDmut) significantly diminished both Gal4 reporter transactivation and Stat5a-mediated gene expression.…”