Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [ Rajagopalan, K. et al. (2014) Biochim, Biophys. Acta 1842, 154-163 ], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.
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