The Stress-response protein prostate-associated gene 4, interacts with c-Jun and potentiates its transactivation

Abstract

The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.

Keywords: BPH; BSA; CD; Cancer/Testis Antigen; DBD; DLS; DMSO; DNA-binding domain; Dimethyl sulfoxide; IPTG; Intrinsically disordered protein; Isopropyl 1-thio-β-d-galactopyranoside; NMR; PAGE4; PBS; PC; PCa; PE; PIA; PIN; PINs; PVP; Phosphate Buffered Saline; Prostate Associated Gene 4; Prostate cancer; QY; SDS-PAGE; SEC; Sodium Dodecyl sulfate poly-acrylamide gel electrophoresis; TCEP·HCl; Tris(2-carboxyethyl)phosphine hydrochloride; benign prostatic hyperplasia; bovine serum albumin; c-Jun; circular dichroism; dynamic light scattering; nuclear magnetic resonance; phosphatidylcholine; phosphatidylethanolamine; polyvinyl pyrollidone; proliferative inflammatory atrophy; prostate cancer; prostatic intraepithelial neoplasia; protein interaction networks; quantum yield; single-molecule Förster resonance energy transfer; size exclusion chromatography; smFRET.

Fig. 1

PAGE4 and c-Jun expression in normal adult and fetal prostate. Messenger RNA levels of PAGE4 (A) and c-Jun (B) were determined in normal adult and fetal prostate by qPCR. Human fetal cDNA was purchased commercially. PAGE4 and c-Jun mRNA expression was normalized to TATA binding protein (TBP). The experiments were repeated three times. Data are represented as mean of triplicate experiments ± SD. (*P < 0.01, Student's t test). (C–E) Fetal prostate was stained for PAGE4 at gestational weeks 12, 21 and 36.

Fig. 2

Single molecule FRET indicates that PAGE4 is an intrinsically disordered protein. (A) Schematic of the PAGE4 constructs with the native cysteine (green) and the introduced cysteine (red). Single PAGE4 protein molecules were encapsulated inside 100 nm diameter liposomes tethered to a quartz surface. (B) Shows a cartoon of this immobilization scheme (not to scale). Fluorescence emission time courses in the donor and acceptor spectral bands were collected and those indicating exactly 1 donor and 1 acceptor were further analyzed. Example intensity timecourses showing anti-correlated donor/acceptor behavior upon photobleaching, which is characteristic of single molecules, are shown for the A18C/63C (C) and P102C/63C (E) FRET mutants. The color bar at the top indicates the illumination color. Red illumination at the start driving only acceptor fluorescence allows identification of molecules containing an active acceptor. The disappearance of red emission (with anticorrelated recovery of green) is photobleaching of the acceptor, and disappearance of green emission is photobleaching of the donor. Histograms assembled from all FRET active data points of over 300molecules are shown for A18C/63C (D) and P102C/63C (F) PAGE4mutants. These FRET signals agree with expectations based upon modeling PAGE4 as a highly flexible IDP.

Fig. 3

PAGE4 potentiates the c-Jun activity. GAL4 (aa 1-147) or GAL4-cJUN (aa 1-223) was cotransfected with or without MEKK and various amounts of V5-tagged PAGE4 (nV5-PAGE4) into PC3 cells. The V5 epitope tag is a small epitope (GKPIPNPLLGLDST) that is derived from the P and V proteins of the paramyxovirus of simian virus 5 and was fused in-frame at the N terminus. Luciferase activities from a GAL4 binding site-driven reporter construct were measured and plotted with reference value normalized to 1.

Fig. 4

Single molecule FRET indicates interaction with c-Jun alters the conformation of PAGE4. Alexa555/Alexa647 labeled PAGE4 containing a 6His tag was directly immobilized on a quartz surface with widely spaced streptavidin molecules adhered, which were used to link to biotinylated-6-His-antibodies. The space between streptavidins was passivated with a lipid bilayer. (A) Displays a cartoon of this immobilization scheme (not to scale). Single molecule fluorescence time traces for donor and acceptor emission were analyzed from molecules confirmed to contain exactly 1 donor and 1 acceptor for the A18C/63C (panel B) and P102C/63C (panel D) FRET mutants in the absence of c-Jun. The color bar at the top indicates the illumination color. Histograms assembled from all FRET active data points of over 300 molecules are shown as the red curves for A18C/63C (panel C) and P102C/63C (panel E) PAGE4 mutants. Note FRET measurements of liposome encapsulated PAGE4 (Fig. 2) agree with the FRET measured from this directly surface immobilized PAGE4. The blue curves are FRET histograms from PAGE4 in the presence of 0.4 mg/ml truncated c-Jun for the A18C/63C (panel C) and P102C/63C (panel E) PAGE4 mutants. C-Jun causes FRET to increase for A18C/63C mutant and to decrease for P102C/63C mutant. The green curves in C and E are for full-length c-Jun.

Fig. 5

Crowding and lipid interactions do not alter PAGE4 conformation. Histograms of single molecule FRET signals from surface immobilized PAGE4 (gray bars) are unchanged from single molecule FRET histograms of surface immobilized PAGE4 in the presence of 0.1 mg/ml bovine serum albumin (red curve) or 20% cardiolipin containing PC liposomes (blue curve) for both the P102C/63C (A) and A18C/63C (B) PAGE4 mutants.

Fig. 6

PAGE4 expression causes down-regulation of p27 in PC3 cells. A luciferase reporter assay was conducted with either wild type (wt) or mutant (mut) p27 3′UTR fused to luciferase. The mut vector had the mir221/222 binding sites scrambled. The assay was done in quadruplicate and the empty vector was normalized to 100%.