title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.
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 androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent 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.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850; herbert.levine@rice.edu pkulkar4@ibbr.umd.edu jorban@umd.edu.
authors.forename: Prakash
authors.initials: P
authors.lastname: Kulkarni
authors.affiliation: Center for Theoretical Biological Physics, Rice University, Houston, TX 77005.
authors.forename: Mohit Kumar
authors.initials: MK
authors.lastname: Jolly
authors.affiliation: Center for Theoretical Biological Physics, Rice University, Houston, TX 77005.|Graduate Program in Systems, Synthetic and Physical Biology, Rice University, Houston, TX 77005.
authors.forename: Dongya
authors.initials: D
authors.lastname: Jia
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, ON Canada N2L 3G1.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Mooney
authors.affiliation: Shakti BioResearch, Woodbridge, CT 06525.
authors.forename: Ajay
authors.initials: A
authors.lastname: Bhargava
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Luciane T
authors.initials: LT
authors.lastname: Kagohara
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Physics, North Carolina State University, Raleigh, NC 27695.
authors.forename: Pengyu
authors.initials: P
authors.lastname: Hao
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850.
authors.forename: Yanan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Veltri
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850.
authors.forename: Alexander
authors.initials: A
authors.lastname: Grishaev
authors.affiliation: Department of Physics, North Carolina State University, Raleigh, NC 27695.
authors.forename: Keith
authors.initials: K
authors.lastname: Weninger
authors.affiliation: Center for Theoretical Biological Physics, Rice University, Houston, TX 77005; herbert.levine@rice.edu pkulkar4@ibbr.umd.edu jorban@umd.edu.|Department of Physics and Astronomy, Rice University, Houston, TX 77005.|Department of Bioengineering, Rice University, Houston, TX 77005.
authors.forename: Herbert
authors.initials: H
authors.lastname: Levine
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850; herbert.levine@rice.edu pkulkar4@ibbr.umd.edu jorban@umd.edu.|Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742.
authors.forename: John
authors.initials: J
authors.lastname: Orban
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010641:Phenotype; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020543:Proteome
chemical_list: D000951:Antigens, Neoplasm; D064267:Intrinsically Disordered Proteins; C474505:PAGE4 protein, human; D020543:Proteome; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases
keywords: PAGE-4; androgen resistance; intrinsic disorder; phenotypic heterogeneity; prostate cancer