Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 1. PAGE4 conformational dynamics and phenotypic heterogeneity in PCa cells. The stress–response kinase HIPK1 phosphorylates PAGE4 at S9 and T51, resulting in a relatively compact PAGE4 ensemble that can potentiate c-Jun (AP-1) in androgen-dependent PCa cells such as LNCaP. In contrast, the dual- specificity kinase CLK2 hyperphosphorylates PAGE4 at eight different S/T resi- dues, 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). Phos- phorylated residues are indicated as solid orange circles.

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 3. Characterization of CLK2-phosphorylated PAGE4 by MALDI mass spectrometry. (A) Mass spectrum of CLK2-phosphorylated PAGE4 obtained by coexpression in E. coli BL21DE3 cells. (B) Mass spectrum of Myc-DDK–tagged PAGE4 isolated by immunoprecipitation from LNCaP cells. a.u., arbitrary units.

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: Mass Spectrometry, Isolation, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 2. CLK2 activity and expression profile compared with HIPK1. (A) CLK2 phosphorylates PAGE4 in vitro. No enzyme control (blue) and the pres- ence of enzyme (red) are shown. (B) Immunoblotting using specific antibodies against HIPK1, CLK2, and beta-actin as described in Materials and Methods. (C) Detection of CLK2 and HIPK1 protein expression in prostate tissue by qIHC in 80 paired cases of PCa and benign adjacent tissue obtained from radical prostatectomies. (Magnification: 20×, 0.5 μm/pixel resolution.)

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: Activity Assay, Expressing, In Vitro, Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 4. PAGE4 phosphorylation sites from NMR. (A) PAGE4 amino acid sequence highlighting the primary HIPK1 phosphorylation site (blue) and CLK2 phosphorylation sites (blue and red). Boxed regions denote key structural features. (B) Two-dimensional 1H-15N HSQC spectrum of HIPK1-PAGE4 with assignments for backbone amides. As- signments in crowded regions are omitted for clarity. (C) Two-dimensional 1H-15N HSQC spectrum of CLK2-PAGE4 with tentative assignments for likely CLK2 phosphorylation sites (red). Circled positions highlight where there is a significant loss of peak intensity relative to HIPK1-PAGE4, indicating CLK2 phosphorylation at that residue.

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: Phospho-proteomics, Sequencing, Residue

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 5. CLK2-PAGE4 attenuates c-Jun transactivation. GAL4-cJun (aa 1–223) was cotransfected with either PAGE4 cDNA alone or with PAGE4- and CLK2-expressing cDNAs into PC3 (human prostate cancer cell line 3) cells. Luciferase (LUC) activity from a GAL4-binding site driving reporter construct was measured as described in the main text. Mock control (lane 1) represents background luminescence, and Luc (lane 2) represents background due to luciferase reporter. Potentiation of c-Jun transactivation in the absence of PAGE4 (lane 3) is compared with potentiation in the presence (lane 4) of PAGE4, and the effect of PAGE4 hyperphosphorylation by CLK2 (CLK2-PAGE4) on c-Jun potentiation is shown in lane 5.

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: Expressing, Luciferase, Activity Assay, Binding Assay, Construct, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity.

doi: 10.1073/pnas.1700082114

Figure Lengend Snippet: Fig. 8. Modeling the PAGE4/AP-1/AR/CLK2 regulatory circuit. (A) Regulatory circuit for PAGE4/AP-1/AR/CLK2 interactions. Dashed red lines denote enzymatic reactions, and solid black lines denote nonenzymatic reactions. CLK2 and HIPK1, the two enzymes involved, are shown in dotted rectangles. (B) Dynamics of the circuit showing sustained and damped oscillations for HIPK1-PAGE4 (PAGE4M, shown in blue), CLK2-PAGE4 (PAGE4H, shown in red), and CLK2 (shown in green). Parameters are given in Fig. S9. (C) Distribution of androgen dependence for an isogenic population over a spectrum, as indicated by the shade of green. Dark green boxes denote highly androgen-dependent (i.e., ADT-sensitive) cells, and white boxes denote androgen-independent cells.

Article Snippet: After overnight incubation at 37 °C in a 5% (vol/vol) CO2, humidified incubator, cells were transfected with 5 μg of Myc-DDK–tagged PAGE4 encoding cDNA (Origene Technologies) using Effectene Transfection Reagent (Qiagen).

Techniques: