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

Published: June 03, 2017

Author(s)

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

Abstract

Intrinsically disordered proteins (IDPs) that lack single fixed 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 AP-1 transcription factor. Homeodomain-interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51 but only T51 is critical for its transcriptional activity. Here, we show CDC-like Kinase 2 (CLK2) to hyperphosphorylate PAGE4 at multiple S/T residues including S9 and T51. Interestingly, cell-based functional studies revealed that while HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiated c-Jun, CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuated it. Small-angle X-ray scattering (SAXS) and single molecule FRET (smFRET) analyses revealed that HIPK1-PAGE4 exhibits a relatively more compact conformation than the native, non-phosphorylated ensemble but the CLK2-PAGE4 ensemble is significantly less compact and more resembling random coil. Paramagnetic relaxation enhancement experiments corroborated the SAXS and smFRET observations and NMR titration experiments showed that while HIPK1 phosphorylation promoted PAGE4/AP-1 interaction, hyperphosphorylation by CLK2 attenuated it agreeing with the in vivo functional studies. While HIPK1 is expressed both in androgen-sensitive and in androgen-resistant prostate cancer (PCa) cells, CLK2, like PAGE4, is expressed only in cells with the former phenotype suggesting that the conformational plasticity of PAGE4 may modulate phenotypic plasticity of PCa cells. We present a mathematical model demonstrating how phosphorylation-induced conformational dynamics of this IDP that remains unstructured even when engaged by its target, can modulate transitions between androgen-sensitive and androgen-resistant phenotypes by rewiring the AP-1/androgen receptor regulatory circuit in PCa cells.
Citation: Proceedings of the National Academy of Sciences of the United States of America
Pub Type: Journals

Keywords

Prostate-associated Gene 4, Homeodomain-interacting protein kinase 1, CDC-like kinase 2, c-Jun, Activator Protein-1, State switching, Cancer/Testis Antigen, bistability, conformational dynamics, intrinsically disordered protein, prostate cancer
Created June 03, 2017, Updated February 07, 2018