Figure 4.
In the M phase, phosphorylation of PC4 at S17 by CK2 hampers its interaction with TRIM28 and subsequent ubiquitination, leading to the degradation of CCND1 mRNA. (A) Western blot showing the interaction between PC4 and CK2 at different cell cycle phases in Huh7 cells. (B) Coomassie staining and western blot showing the phosphorylation of PC4 in vitro phosphorylation assay with the recombinant proteins GST-PC4, CK2, and CK2 inhibitor CX-4945. (C) Western blot showing the S17 phosphorylation of PC4 in Huh7 cells with stable PC4 knockdown and re-expression of Flag-tagged PC4WT upon CK2 inhibitor CX-4945 treatment. (D) Coomassie staining and western blot showing S17 phosphorylation of PC4 in vitro phosphorylation assay with the recombinant CK2 and GST-PC4 variants proteins. (E) Western blot showing the ubiquitination of PC4 in Huh7 cells transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 knockdown. EV, empty vector; IB, immunoblot. (F) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants. (G) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 overexpression. (H) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 inhibitor CX-4945. (I) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in phosphorylation assay and ubiquitination assay with recombinant proteins GST-PC4, GST-PC4S17A and GST-PC4S17E, followed by GST pull-down. * represents phosphorylated GST-tagged recombinant PC4. ^ represents non-phosphorylated GST-tagged recombinant PC4. (J) RIP-qPCR and western blot showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells. Data were generated from n = 3 biological replicates. (K) In vitro EMSA analysis showing the interaction between GST-tagged recombinant PC4 variant proteins and CCND1 5′UTR after phosphorylation assay and ubiquitination assay. (L) RIP-qPCR showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells at different cell cycle phases. Data were generated from n = 3 biological replicates. (M) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-depleted Huh7 cells stably expressing indicated Flag-tagged PC4 variants and CK2 at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. A–I are representative of three independent experiments. Source data are available for this figure: SourceData F4.

In the M phase, phosphorylation of PC4 at S17 by CK2 hampers its interaction with TRIM28 and subsequent ubiquitination, leading to the degradation of CCND1 mRNA. (A) Western blot showing the interaction between PC4 and CK2 at different cell cycle phases in Huh7 cells. (B) Coomassie staining and western blot showing the phosphorylation of PC4 in vitro phosphorylation assay with the recombinant proteins GST-PC4, CK2, and CK2 inhibitor CX-4945. (C) Western blot showing the S17 phosphorylation of PC4 in Huh7 cells with stable PC4 knockdown and re-expression of Flag-tagged PC4WT upon CK2 inhibitor CX-4945 treatment. (D) Coomassie staining and western blot showing S17 phosphorylation of PC4 in vitro phosphorylation assay with the recombinant CK2 and GST-PC4 variants proteins. (E) Western blot showing the ubiquitination of PC4 in Huh7 cells transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 knockdown. EV, empty vector; IB, immunoblot. (F) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants. (G) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 overexpression. (H) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in Huh7 transfected with K63-linked HA-tagged ubiquitin and indicated Flag-tagged PC4 variants with or without CK2 inhibitor CX-4945. (I) Western blot showing the ubiquitination of PC4 and the interaction of PC4 and TRIM28 in phosphorylation assay and ubiquitination assay with recombinant proteins GST-PC4, GST-PC4S17A and GST-PC4S17E, followed by GST pull-down. * represents phosphorylated GST-tagged recombinant PC4. ^ represents non-phosphorylated GST-tagged recombinant PC4. (J) RIP-qPCR and western blot showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells. Data were generated from n = 3 biological replicates. (K) In vitro EMSA analysis showing the interaction between GST-tagged recombinant PC4 variant proteins and CCND1 5′UTR after phosphorylation assay and ubiquitination assay. (L) RIP-qPCR showing the association of indicated Flag-tagged PC4 variants and CCND1 mRNA in Huh7 cells at different cell cycle phases. Data were generated from n = 3 biological replicates. (M) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-depleted Huh7 cells stably expressing indicated Flag-tagged PC4 variants and CK2 at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. A–I are representative of three independent experiments. Source data are available for this figure: SourceData F4.

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