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.

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

“…FRET measurements repeated here had FRET efficiency of 0.55 (A18:C63) and 0.65 (C63:P102) for WT-PAGE4 and FRET efficiency of 0.52 (A18: C63) and 0.65 (C63:P102) for HIPK1-PAGE4. These smFRET values were consistent with our previous studies (32,37). Using the same labeling sites with CLK2-PAGE4, we found FRET efficiency of 0.35 (A18:C63) and 0.58 (C63:P102).…”

“…We had previously observed that PAGE4 phosphorylated by HIPK1 prefers a more compact conformation relative to the WT PAGE4 ensemble (37) and potentiates transactivation of c-Jun in a cell-based transactivation assay using a luciferase reporter (32). Therefore, we determined the effect of hyperphosphorylation of PAGE4 on c-Jun potentiation using the same luciferase reporter assay in PC3 cells that were cotransfected with full-length human CLK2 cDNA.…”

“…It has the hallmarks of a protooncogene, with important roles in development and differentiation. Thus, the protein is highly expressed in the human fetal prostate, trophoblasts, and placenta (31,32), as well as in reprogrammed induced pluripotent stem cells, but it is undetectable in the parent somatic cells used for reprogramming (reviewed in ref. 33).…”

“…However, PAGE4 protein expression is elevated in prostate cancer (PCa) (31,(34)(35)(36), where it is upregulated in response to a variety of stress factors (29,31). PAGE4 is phosphorylated at S9 and T51 by HomeodomainInteracting Protein Kinase 1 (HIPK1), a component of the cellular stress-response pathway, and functions as a strong potentiator of the oncoprotein c-Jun (32,37). c-Jun is also a component of the stress-response pathway, and forms a heterodimer with members of the Fos family to produce Activator Protein-1 (AP-1).…”

“…Potentiation of c-Jun by PAGE4 was determined using the luciferase reporter assay as described previously (32). Briefly, PC3 cells (8,000 cells per well) were seeded in 100 μL of RPMI/10% (vol/vol) FBS/PS (penicillin, streptomycin mix) in a 96-well plate and incubated overnight at 37°C in a humidified, 5% (vol/vol) CO 2 incubator.…”