Fig 2: T410 phosphorylation of RIOK1 antagonists SETD7-mediated K411 methylation, which stabilizes RIOK1 in CRC and GC cells.(A) The effect of knockdown of CK2a in HCT116 cells on the levels of RIOK1 proteins, T410p, and K411me. (B) The effect of the CK2a-K68A mutant on the levels of RIOK1 proteins, T410p, and K411me. (C) The effect of the CK2 kinase inhibitor TBB on the levels of RIOK1 proteins, T410p, and K411me. (D) The effect of CK2 knockdown in HCT116 cells on the RIOK1 protein stability. (E) CK2 inhibits K411me by SETD7 and its consequent RIOK1 ubiquitination in RKO cells. Indicated cells were cotransfected with HA-Ub and treated with MG132 for 12 hr. (F) Wild-type RIOK1 or the RIOK1-K411me1 mutant was overexpressed in cells, along with HA-ubiquitin and V5-SET7, in the presence or absence of the proteasome inhibitor MG132. Cell lysates were analyzed with indicated antibodies.

Fig 3: CK2 phosphorylates LSD1 at S131 and S137 both in vitro and in vivo. (A and B) Endogenous CK2a’ co-immunoprecipitated reciprocally with endogenous LSD1. Total cell lysates were extracted from 293T cells and subjected to immunoprecipitation followed by immunoblotting with antibodies as indicated. (C) LSD1 directly interacted with CK2a’. GST or GST- CK2a’ recombinant protein was employed to pull down bacterially produced HIS-LSD1. (D) CK2 phosphorylated LSD1 in vitro. GST or GST-CK2a’ recombinant protein was incubated with bacterially produced GST-LSD1 or its phosphorylation-defective mutants in the presence of 32P-?ATP in the in vitro kinase assay. (E) The phospho-specific antibodies were reactive toward LSD1 phosphorylated by CK2 in vitro. Bacterially produced GST-LSD1 was incubated with GST-CK2 and subjected to immunoblotting with antibodies as indicated. (F) Specificity of the LSD1 phospho-specific antibodies. Total cell lysates were extracted from 293T cells transiently transfected with HA-Vec or expression constructs as indicated, and the HA immunocomplexes were then subjected to immunoblotting with antibodies as indicated. (G) Inhibition of CK2 expression diminished LSD1 phosphorylation at S131. Total cell lysates were extracted from HeLa cells with mock depletion or depletion of both CK2a and CK2a’ and subjected to immunoblotting with antibodies as indicated.

Fig 4: LSD1 phosphorylation promotes the interaction between 53BP1 and RNF168 in chromatin. (A) LSD1 interacted directly with 53BP1 and CK2-mediated phosphorylation promoted this interaction. Bacterially produced GST-53BP1-KBD was used to pull down bacterially produced HIS-RNF168 in the absence of HIS-LSD1 or in the presence of HIS-LSD1, HIS-LSD1 phosphorylated by CK2a’ in vitro in the absence/presence of the CK2 inhibitor TBB. (B) Inhibition of LSD1 expression compromised the interaction between 53BP1 and RNF168 in the chromatin-enriched fraction. 293T cells were transfected first with siCTR or siLSD1 and then with FLAG-RNF168 24 h after the first transfection. Cells were treated with bleomycin (10 µg/ml) for 1 h before harvest. Chromatin-enriched fractions (P3) were extracted 48 h after the second transfection and subjected to immunoprecipitation followed by immunoblotting with antibodies as indicated. (C) Phosphorylation-defective LSD1 mutants reduced the interaction between 53BP1 and RNF168. 293T cells were transfected first with siCTR or siLSD1 and then co-transfected with FLAG-RNF168 and HA-Vec/HA-LSD1/HA-LSD1(S131A)/HA-LSD1(S137A)/HA-LSD1(2A) 24 h after the first transfection. Chromatin-enriched fractions (P3) were extracted 48 h after the second transfection and subjected to immunoprecipitation with an anti-53BP1 antibody followed by immunoblotting with antibodies as indicated. (D) Inhibition of LSD1 expression compromised RNF168-mediated 53BP1 ubiquitination. 293T cells were transfected first with siCTR or siLSD1 and then co-transfected with HA-Vec/HA-UB(K63) and MYC-Vec/MYC-RNF168 24 h after the first transfection. Total cell lysates were extracted 48 h after the second transfection and subjected to immunoprecipitation with an anti-53BP1 antibody followed by immunoblotting with antibodies as indicated.

Fig 5: CK2-mediated phosphorylation of LSD1 promotes its association with RNF168. (A) LSD1 directly interacted with RNF168. Bacterially produced LSD1 was used to pull down bacterially produced HIS-RNF168 in vitro. (B) CK2-mediated phosphorylation of LSD1 enhanced its binding to RNF168 in vitro. Bacterially produced GST, GST-LSD1 and GST-LSD1 phosphorylated by CK2 in vitro in the absence/presence of the CK2 inhibitor TBB were employed to pull down bacterially produced HIS-RNF168. (C) Phosphorylation-defective mutants of LSD1 reduced their binding to RNF168 in vivo. 293T cells were transiently co-transfected with FLAG-RNF168 and HA-Vec/HA-LSD1/HA-LSD1(S131A)/HA-LSD1(S137A)/HA-LSD1(2A). Total cell lysates were extracted 48 h after transfection and subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with antibodies as indicated. (D) Inhibition of CK2 activity in vivo reduced the binding of LSD1 to RNF168. 293T cells were co-transfected with FLAG-RNF168 and HA-Vec/HA-LSD1. Transfectants were treated with different concentrations of TBB 6 h before harvest. Total cell lysates were harvested 48 h after transfection and subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with antibodies as indicated. (E) Bleomycin treatment increased LSD1 phosphorylation in the chromatin-enriched fraction. 293T cells were transiently transfected with HA-Vec or HA-LSD1. HA-LSD1 transfectants were either mock-treated or treated with bleomycin (10 µg/ml) for 1 h before harvest. Chromatin-enriched fractions (P3) were prepared 48 h after transfection and subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with antibodies as indicated. (F) DNA damage induced an increase of interaction between RNF168 and LSD1 in the chromatin-enriched fraction. 293T cells were transiently co-transfected with FLAG-RNF168 and HA-Vec/HA-LSD1. Co-transfectants with FLAG-RNF168 and HA-LSD1 were either mock-treated or treated with bleomycin (10 µg/ml) for different time points as indicated before harvest. Chromatin-enriched fractions (P3) were prepared 48 h after transfection and subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with antibodies as indicated.