Cancer/Testis Antigen PAGE4, a Regulator of c-Jun Transactivation, Is Phosphorylated by Homeodomain-Interacting Protein Kinase 1, a Component of the Stress-Response Pathway

Abstract

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 [RajagopalanK.RajagopalanK.24263171Biochim, Biophys. Acta20141842154], 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.

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

“…Thus, the population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on T51. Further, consistent with our single-molecule fluorescence resonance energy transfer (smFRET) microscopy results (37), this central region becomes more compact and more negatively charged upon phosphorylation with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Furthermore, we observed that although flexibility is decreased in the central region of the phosphorylated ensemble, the polypeptide chain remains disordered and highly dynamic overall.…”

“…2B), HIPK1 protein was detected at almost the same level in BPH-1 cells and in the LNCaP cells that are androgen-dependent and nonaggressive, as well as in the androgen-independent and aggressive DU145 and PC3 cells. In contrast, CLK2 was expressed only in In contrast, the dualspecificity kinase CLK2 hyperphosphorylates PAGE4 at eight different S/T residues, including S9 and S51, leading to a more random-like PAGE4 ensemble that attenuates c-Jun transactivation and is likely to be degraded rapidly (9,37,77). Differential phosphorylation of PAGE4 by HIPK1 and CLK2 results in oscillations of the levels of HIPK1-PAGE4, CLK2-PAGE4, and CLK2 that can be modeled mathematically to correlate with the experimentally observed heterogeneity in a population of isogenic PCa cells (details are provided in the main text).…”

“…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.…”

“…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).…”

“…This group of early response transcription factors represents a paradigm for signal-responsive factors with important roles in the control of cell growth, apoptosis, and stress response (38). Phosphorylation at T51 is necessary because mutation of this residue to an alanine abolishes its activity by >90% (37).…”