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

Prakash Kulkarni, Mohit Kumar Jolly, Dongya Jia, Steven M. Mooney, Ajay Bhargava, Luciane T. Kagohara, Yihong Chen, Pengyu Hao, Yanan He, Robert W. Veltri, Alexander Grishaev, Keith Weninger, Herbert Levine, John Orban

Research output: Contribution to journalArticle

Abstract

Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgendependent and androgen-independent prostate cancer (PCA) cells, whereas CLK2 and PAGE4 are expressed only in androgendependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun,whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-Angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCA cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCA cells.

Original languageEnglish (US)
Pages (from-to)E2644-E2653
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number13
DOIs
StatePublished - Mar 28 2017

Keywords

  • Androgen resistance
  • Intrinsic disorder
  • Page-4
  • Phenotypic heterogeneity
  • Prostate cancer

ASJC Scopus subject areas

  • General

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    Kulkarni, P., Jolly, M. K., Jia, D., Mooney, S. M., Bhargava, A., Kagohara, L. T., Chen, Y., Hao, P., He, Y., Veltri, R. W., Grishaev, A., Weninger, K., Levine, H., & Orban, J. (2017). Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity. Proceedings of the National Academy of Sciences of the United States of America, 114(13), E2644-E2653. https://doi.org/10.1073/pnas.1700082114/-/DCSupplemental