Fig 2: In vitro phosphorylation of Rad9 C-terminal fragments by TLK1.A & B. Recombinant GST-fusion peptides corresponding to different regions of Rad9’s C-terminal tail were employed as substrates for in vitro kinase assays using TLK1 immunoprecipitated from HeLa cells exposed to the indicated conditions. 3A refers to a T355A/S363A/S387A triple mutant. C. Substrate input (Rad9, left panel) and kinase input (TLK1, right panel) for each kinase reaction. IP refers to immunoprecipitate, IB refers to immunoblot. D. Overexpressed, recombinant WT and kinase-dead (D607A) myc-TLK1 constructs were immunoprecipitated from HeLa cells and employed in in vitro kinase assays using full-length GST-Rad9 and GST-ASF1a as substrates. E. A time course in vitro kinase assay. TLK1 was immunoprecipitated from HeLa cells exposed to 20Gy IR and harvested at the indicated time-points. Immune complexes were incubated with either recombinant GST-Rad9 (amino acids 348-391) or GST- ASF1A. SCL refers to soluble cell lysates. Images shown are representative of two independent experiments.

Fig 3: The forced expression of Tlk1 results in the aberrant downregulation of core pluripotency factors and attenuates self-renewal. (A) Immunoblot analysis of Oct4, Sox2, and Nanog levels in control mESCs (empty vector or doxycycline depletion) and Tlk1-overexpressing mESCs. The mESCs expressing an empty vector or the Tet-On-Tlk1 or Tet-On-Tlk1-D607A expression vector were cultured in the absence or presence of doxycycline (Dox; 100 ng/ml) for 24 hrs under undifferentiated self-renewal conditions. (B) Quantification of results from (A). The protein levels of the target genes were normalized to a-tubulin levels. The protein expression levels of each mESC line not treated with doxycycline were normalized to 1. The biological data are presented as mean (n = 6) ± SEM. *? < 0.05, and **P < 0.01. (C) The morphology and AP staining of Tet-On-inducible Tlk1-expressing cell lines cultured in mock (Dox-) or doxycycline (Dox+) for 48 hrs. Scale bar, 500 µm.

Fig 4: Tlk1 deficiency leads to a failure in the scheduled downregulation of pluripotency-associated genes and the aberrant expression of lineage-associated genes. (A) The KD efficiency of Tlk1 in control (shLuc) and Tlk1-KD (shTlk1 #1 and #2) mESCs cultured in three separate differentiation conditions was confirmed by RT-qPCR. Differentiation was induced by LIF withdrawal (LIF-), embryoid body formation (EB), and by retinoic acid treatment (RA). (B–D) The mRNA expression of pluripotency-associated and development-associated genes under three separate differentiation conditions including LIF withdrawal (B), embryoid body formation (C), and retinoic acid treatment (D) was analyzed by RT-qPCR. All data are normalized to Gapdh. The mRNA levels in luciferase (Luc)-KD control cells were normalized to 1. Data are means (n = 3) ± SEM for LIF- and EB and for RA (n = 4). *Р < 0.05, **Р < 0.01, and ***Р < 0.001.

Fig 5: Architecture and characterization of TLK2. a TLK2 domain architecture and constructs used in this study. For a complete list see Supplementary Figure 2. b Size exclusion chromatography (SEC) coupled with Multi-Angle Laser Light Scattering (MALLS) profiles were used to assess the oligomerization state of the phosphorylated and unphosphorylated constructs with or without the C-tail for the kinase domain and c ΔN-TLK2 constructs. The effect of the catalytically inactive mutant ΔN-TLK-KD (D613A mutation) is also included. d The indicated TLK2 mutants were overexpressed in AD293 cells by transient transfection and pulled down from cell lysates using Streptavidin resin. Protein–protein interactions were analyzed by western blotting for TLK2, TLK1, ASF1 and LC8 (right panel). Input levels are shown in the left panel including the Ponceau red-stained membrane showing similar total protein levels. The KD in this experiment bears the D592V mutation instead of D613A. e Kinase assays were performed from Streptavidin pull-downs performed as in d. Kinase complexes were incubated with either MBP or ASF1a in the presence of 32P-ATP and autophosphorylation (TLK2) and substrate phosphorylation assessed in dried SDS-PAGE gels exposed to a phosphoimager. f Immunofluorescence analysis of exogenous TLK2 in transiently transfected AD293 cells. Localization of TLK2 and mutant forms was determined by staining with anti-FLAG antibodies and co-staining of nuclei with DAPI. Cytoplasmic localization of the ∆N-TLK2 mutant confirms the presence of the NLS in the N-terminus of the protein. The scale bar represents 10 μm in all the pictures. Uncropped gels and blots are shown in Supplementary Figures 10–15