Abstract
Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985-13994). Phosphorylation of PAGE4 at Thr-51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable ("metamorphic") folded proteins in response to mutation or environmental triggers.
Highlights
Prostate-associated gene 4 (PAGE4), an intrinsically disordered protein up-regulated in prostate cancer, binds to c-Jun and potentiates its transactivation
Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51
Sample Preparation and NMR Assignments—WT-PAGE4 samples were prepared using the eXact tag purification system, which has the advantage over most other methods of providing the desired naturally occurring polypeptide chain with no additional residues at either end [32]
Summary
PAGE4, an intrinsically disordered protein up-regulated in prostate cancer, binds to c-Jun and potentiates its transactivation. Significance: This study may help to explain how phosphorylation of PAGE4 alters its binding to c-Jun. Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Prostate-associated gene 4 (PAGE4) is a 102-amino acid member of the PAGE4 family of cancer/testis antigens, a heterogeneous group of proteins typically expressed in testicular germ cells. The PAGE4 protein is up-regulated in both symptomatic benign prostatic hyperplasia, which is related to prostate cancer [13], and in prostate cancer itself [12, 15, 16], indicating a potential role for PAGE4 in disease pathology Consistent with this hypothesis, knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells [17]. This is similar conceptually to the expanded function observed for both naturally occurring and designed proteins that can switch their fold topologies [30, 31]
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