Fig 2: Casein kinase II phosphorylation regulates SCF(cyclin F) Lys48-specific E3 ligase activity. (a) E3 ligase activity of Lys48-ubiquitylation from immunoprecipitated mCherry–cyclin F in transfected cells treated with CX4945 (4 µM) demonstrated elevated ubiquitylation activity with CK2 inhibition by approximately 1.35-fold (p = 0.0215, n = 3, one-way ANOVA). Immunoblots were carried out following E3 ligase activity assays to demonstrate equivalent levels of immunoprecipitated mCherry–cyclin F used for activity assays, which also revealed cyclin F contained a CK2 phosphorylation motif. (b) Immunoblot analysis of phospho-cyclin F (Ser621) using custom antibodies revealed reduced phosphorylation by approximately 0.68-fold (p = 0.0154, n = 3, one-way ANOVA) with CX4945 treatment. Vertical dashed line indicates cropped lanes. (c) E3 ligase activity of Lys48-ubiquitylation from immunoprecipitated mCherry–cyclin F in cells co-transfected with mCherry–CCNFWT or mCherry–CCNFS621G with scramble or CK2 siRNA demonstrated elevated ubiquitylation activity with CK2 knock down by approximately 1.2-fold (p = 0.0136, n = 3, one-way ANOVA). Immunoblots were carried out following E3 ligase activity assays to demonstrate equivalent levels of immunoprecipitated mCherry–cyclin F used for activity assays. (d) Immunoblot analysis of phospho-cyclin F (Ser621) using custom antibodies revealed reduced phosphorylation by approximately 0.61-fold (p = 0.005, n = 3, one-way ANOVA) with CK2 RNAi knockdown. Data are represented as the mean ± s.e.m. using one-way ANOVA with Tukey's post hoc test.

Fig 3: NS2 phosphorylation at Ser-249 and Ser-259 decreases following CK2 inhibition or PP2A activation. HeLa cells infected with BTV1 (MOI = 1) were treated 20 h.p.i. for 4 h with 100 µm TBB, 10 µm FTY720, and 20 nm okadaic acid. NS2 was isolated from these cells using immunoprecipitation (IP). A, samples were analyzed by Western blotting to confirm NS2 identity. SDS-PAGE gels were sequentially stained for phosphoproteins and total proteins. B, densitometry analysis of phosphoprotein and total protein staining data in A from replicate experiments. NS2 is indicated along the detected Ig heavy chains (HC). Error bars represent the standard deviation values of stimulations from three independent experiments. *, p < 0.05.

Fig 4: CK2 activity is important for viral replication and for regulating VIB morphology during BTV infection. HeLa cells infected with BTV1 (MOI = 1) were treated 4 h.p.i. with concentrations of 100, 50, 20, and 10 µm TBB for 20 h. A, samples were analyzed by Western blotting using specific antibodies as indicated. B, densitometry analysis of the Western blots expressed as a percentage. C, virus titer derived from HeLa cells infected with BTV1 (MOI = 1) and treated 4 h.p.i. with concentrations of 100, 50, 20, and 10 µm TBB for 20 h. D, HeLa cells infected with BTV1 (MOI = 1) were treated 20 h.p.i. with concentrations of 100, 50, 20, and 10 µm TBB for 4 h. E, VIBs denoted by NS2 staining were quantified using Volocity software, and their size was determined in replicate experiments of D. Error bars represent the standard deviation (Western blots) and standard error (confocal images) values of stimulations from three independent experiments. *, p < 0.05.

Fig 5: Proposed mechanism of phosphorylated Ser621 in cyclin F and its effect on the SCF(cyclin F) Lys48-ubiquitylation activity. CK2 is responsible for phosphorylating Ser621 in cyclin F and regulating the SCF(cyclin F) E3 ligase activity. Inhibition of CK2 activity by either CX4945 or siRNA knock down prevents phosphorylation of mCherry–cyclin F at Ser621 and elevates its Lys48-specific E3 ligase activity. Additionally, a mutation at Ser621 (p.Ser621Gly) has been found in a large multi-generational Australian family with ALS/FTD, and expression of this mutant version (cyclin FS621G) in cells displays elevated Lys48-specific E3 ligase activity.