Fig 2: Human CHEK2 variants and their effect on CHK2 expression and kinase activity toward Kap1 p.S473. Western blot analysis of the indicated proteins from Chek2KO mES cells expressing wild-type (WT; black) human untagged CHK2, empty vector (Ev; gray), or the indicated untagged CHK2 variants in untreated conditions (no IR) or at 2 or 6 hours after IR exposure (10 Gy). WT and Ev served as controls on each blot and variants are categorized by color as either synonymous (green), truncating (red), and missense VUS (blue). Tubulin was used as a loading control. Dashed lines represent a marking of different set of samples on the same blot, whereas continuous lines are used to mark different sets of samples from distinct and separately exposed blots.

Fig 3: MLKL oligomerization induces p38 activation to mediate the phosphorylation of Ser473 TRIM28.A HT-29 cells were treated with the indicated inhibitors followed by TSZ treatment for 4 h. The cell lysates were analyzed by western blotting using indicated antibodies. B p38 KO cells were engineered by CRISPR–Cas9 technology and p38 expressing vector was reconstituted in HT-29 cells. WT and p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s at indicated time points. The cell lysates were analyzed by western blotting using indicated antibodies. C WT and p38 reconstituted p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s as indicated. The cell lysates were analyzed by western blotting using indicated antibodies. D, E HT-29 cells were transfected with an expression vector of flag-tagged MLKL fused with two AP20187-binding (FKBPv) domains. The cells were pretreated with indicated compounds for 4 h followed by treatment with AP20187 to induce MLKL oligomerization. Cell viability was determined by CellTiter-Glo after treatment with AP20187 for 2 h (D Left). Cell lysates were separated by non-reducing SDS/PAGE and analyzed by western blotting using MLKL antibody (D Right). The cell lysates were immunoprecipitated by anti-Flag resin after treatment with AP20187 for 2 h and analyzed by western blotting using indicated antibodies (E). acMLKL, oligomerizable MLKL; endo, endogenous. TNFa, 20 ng/mL; zVAD, 25 µM; SM164, 50 nM; Nec-1s, 10 µM; NSA, 4 µM; TPCA-1, 10 µM; p38i PH-797804, 10 µM; p38i LY2228820, 4 µM; 5Z-7, 500 nM; MK2i MK2-IN-1, 10 µM; AP20187, 20 µM. Data were presented as mean ± SEM. **t-test p < 0.01, n = 3. n.s., not significant.

Fig 4: Correlation between computational predictions and functionality of CHEK2 variants. A, Bar plot showing the R2-correlation values between the FACS-based analysis of Kap1 p.S473 phosphorylation as shown in Fig. 3C and computational predictions from 12 different prediction algorithms. B, Scatter plot showing the correlation between Helix-based in silico predictions and results from functional assays presented in our study (Fig. 3C), or those from Delimitsou and colleagues 2019 (34) and Kleiblova and colleagues 2019 (35). Data points are colored on the basis of functional classification (green, functional; orange, intermediate; red, damaging). Helix provides predictions for pathogenicity ranging from 0–1, with values close to 1 representing pathogenic predictions. C, En masse prediction plot from Helix for all possible missense changes in human CHEK2. Schematic representation of the CHK2 protein and its functional domains demarcated by the amino acid numbers at the x-axis of the plot. D, Heatmap showing predictions from Helix combined with functional data for CHK2 amino acid changes that were analyzed in Fig. 3C (outlined in bold). Functional variants are indicated in green (with bold outline); amino acid changes with a similar (+0.05) or lower prediction from Helix are also indicated in green. Intermediate variants are indicated in orange (with bold outline); amino acid changes with a similar (-0.05) or higher prediction from Helix are also indicated in orange. Damaging variants are indicated in red (with bold outline); amino acid changes with a similar (-0.05) or higher prediction from Helix are also indicated in red. For each amino acid position, amino acid changes with a similar color code are expected to result in similar functional effects. Squares in gray and white represent changes into the original amino acid or variant changes for which predictions are unclear, respectively.

Fig 5: TRIM28 interacts with MLKL and is phosphorylated at Ser473 in complex IIb during necroptosis.A, B HT-29 cells were treated with the compounds as indicated. The levels of specific proteins were analyzed by western blotting using indicated antibodies. C, D RIPK1−/− (C) and MLKL−/− (D) MEFs were treated with TSZ as indicated. The levels of total TRIM28 and phosphorylation of TRIM28 S473 were analyzed by western blotting. E HT-29 cells were treated with TSZ or TSZ + Nec-1s for 4 h. Lysates were immunoprecipitated with MLKL antibody and analyzed by western blotting using indicated antibodies. F MEFs were treated with TSZ for 3 h. The cell lysates were immunoprecipitated by pRIPK1S166 antibody. Both lysate input and immunoprecipitation samples were analyzed by western blotting using indicated antibodies. *, non-specific bands. G 293T cells were transfected with GFP-MLKL and HA-TRIM28 for 24 h. The cell lysates were immunoprecipitated with anti-HA resin and analyzed by western blotting using indicated antibodies. H 293T cells were transfected with tet-inducible flag-tagged MLKL Q356A expression vector. MLKL oligomerization was induced by doxycycline with or without NSA. Cell viability was determined by CellTiter-Glo (left). Cell lysates were separated by non-reducing SDS/PAGE and analyzed by western blotting using MLKL antibody (right). I 293T cells were transfected with tet-inducible flag-tagged MLKL Q356A expression vector. After doxycycline induction, the cell lysates were prepared and immunoprecipitated by anti-Flag resin and analyzed by western blotting using indicating antibodies. TNFα, 20 ng/mL; zVAD, 25 μM; SM164, 50 nM; Nec-1s, 10 μM; GSK’872, 10 μM; NSA, 2 μM; Dox, 2 ng/mL. Data were presented as mean ± SEM. ***t-test p < 0.001, n = 3.