Fig. 6: CK2 phosphorylates ARKL1 promoting ARKL1-RNF111 interaction and BLM ubiquitination. | Nature Communications

Fig. 6: CK2 phosphorylates ARKL1 promoting ARKL1-RNF111 interaction and BLM ubiquitination.

From: A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution

Fig. 6

a CK2 inhibitor TBB treatment abolishes HA-RNF111 localization to PML NBs. Immunofluorescence (IF) in RNF111 KO U2OS cells expressing HA-RNF111 was quantified for DMSO-treated (n = 101) or TBB (50 μM, 4 h)-treated (n = 100) cells from two biological replicates. b PLA in RNF111 KO cells expressing HA-RNF111 was quantified for DMSO-treated (n = 43) or TBB-treated (n = 37) cells. c TBB treatment reduces BLM ubiquitination. d TBB treatment leads to increased BLM levels in PML NBs. IF was quantified with arbitrary (arb.) units for DMSO-treated (n = 1354) or TBB-treated (n = 903) cells. e The serine-rich region of ARKL1 is required for promoting RNF111-PML colocalization. PLA was quantified for Hela WT expressing vector (n = 152) or ARKL1 KO expressing vector (n = 178), ARKL1WT (n = 180), or ARKL1ΔS (n = 130) from two biological replicates. Expression of the indicated proteins is shown in Supplementary Fig. 6c. f ARKL1WT but not ARKL1ΔS or ARKL1SIM* mutant reduces the elevated BLM levels in PML NBs in ARKL1 KO cells. Quantifications are shown for U2OS WT expressing vector (n = 1302) or ARKL1 KO cells expressing vector (n = 2109), ARKL1WT (n = 1467), ARKL1ΔS (n = 2307), ARKL1SIM (n = 3015). Expression of the indicated proteins is shown in Supplementary Fig. 6d. g TBB treatment leads to decreased CK2 phosphorylation of ARKL1. The IPs were performed under denature condition. h ARKL1ΔS mutant cannot be phosphorylated by CK2. Hela cells expressing indicated constructs were used. i Identification of CK2 phosphorylation site on ARKL1. 293T cells expressing vector, HA-tagged ARKL1 WT, S328A or 4 A (S327/328/330/385 A) mutant were used. j TBB treatment reduces the interaction of ARKL1-Flag with RNF111. k ARKL1ΔS and ARKL1S328A mutants exhibit decreased interaction with RNF111. Quantifications are shown with mean ± 95% CI for a, b, d–f. Statistics: two-tailed unpaired t-test for a–d, one-way ANOVA with Sidak’s correction for e, f. Scale bar, 10 μm for a, b, d–f. Source data are provided as a Source Data file.

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