Figure 3.
Figure 3. PPM1D plays a central role in the DDR pathway. (A) Whole-cell lysates of Molm13 PPM1D-mutant and Molm13 isogenic control single-cell clones exposed to 400 nM cytarabine for 4 hours were probed with anti-p53 Ser15 and anti-CHEK1 Ser345. (B) The log fold-change of different peptides for Molm13 PPM1D-mutant cells compared with Molm13 control cells. The starting amino acid for each peptide is shown. The peptides range from 8 to 22 aa in length. (C) Heatmap of phosphosites that are significantly downregulated with an FDR < 0.05 in Molm13 PPM1D-mutant/control at baseline (Mut/cntrl) or after cytarabine (AraC) treatment (Mut AraC/cntrl AraC), and upregulated (FDR < 0.05) after 4 hours of treatment with 400 nM cytarabine and GSK2830371 (PPM1D inhibitor) (Mut AraC + PPM1Di/Mut AraC). Results shown are for biological replicates. (D) Heatmap of phosphosites belonging to the KEGG_P53 pathway that are significantly regulated (FDR < 0.1) in Molm13 PPM1D-mutant/control after 4-hour treatment with 400 nM cytarabine (AraC) (Mut AraC/cntrl AraC) or in Molm13 PPM1D-mutant cells treated with 400 nM AraC + 1 μM PPM1D inhibitor GSK2830371 compared with Molm13 PPM1D mut cells treated with 400nM AraC (Mut AraC + iPPM1D/Mut AraC) for 4 hours. Results shown are for biological replicates. (E) Conserved amino acid residues flanking PPM1D-dependent phosphorylation sites based on 43 substrate candidates that are downregulated (FDR < 0.1) in Molm13 PPM1D-mutant/cntrl for either the baseline or the AraC-treated comparisons and at the same time upregulated (FDR < 0.1) after PPM1D inhibitor GSK2830371 treatment. The combined occurrence of glutamine at +1 and ≥2 acidic residues in the flanking region is statistically significant (P = 3.595e-13, Fisher exact test). (F) Schema illustrating the components of the DDR pathway that are targeted by PPM1D in leukemia cells, based on the results from the phosphoproteomic analysis. Phosphorylation targets of PPM1D identified by mass spectrometry (FDR < 0.1) are shown in green. Predicted PPM1D target sites that were based on the identified consensus sequence with a glutamine at +1 and ≥2 acidic residues are shown in blue.

PPM1D plays a central role in the DDR pathway. (A) Whole-cell lysates of Molm13 PPM1D-mutant and Molm13 isogenic control single-cell clones exposed to 400 nM cytarabine for 4 hours were probed with anti-p53 Ser15 and anti-CHEK1 Ser345. (B) The log fold-change of different peptides for Molm13 PPM1D-mutant cells compared with Molm13 control cells. The starting amino acid for each peptide is shown. The peptides range from 8 to 22 aa in length. (C) Heatmap of phosphosites that are significantly downregulated with an FDR < 0.05 in Molm13 PPM1D-mutant/control at baseline (Mut/cntrl) or after cytarabine (AraC) treatment (Mut AraC/cntrl AraC), and upregulated (FDR < 0.05) after 4 hours of treatment with 400 nM cytarabine and GSK2830371 (PPM1D inhibitor) (Mut AraC + PPM1Di/Mut AraC). Results shown are for biological replicates. (D) Heatmap of phosphosites belonging to the KEGG_P53 pathway that are significantly regulated (FDR < 0.1) in Molm13 PPM1D-mutant/control after 4-hour treatment with 400 nM cytarabine (AraC) (Mut AraC/cntrl AraC) or in Molm13 PPM1D-mutant cells treated with 400 nM AraC + 1 μM PPM1D inhibitor GSK2830371 compared with Molm13 PPM1D mut cells treated with 400nM AraC (Mut AraC + iPPM1D/Mut AraC) for 4 hours. Results shown are for biological replicates. (E) Conserved amino acid residues flanking PPM1D-dependent phosphorylation sites based on 43 substrate candidates that are downregulated (FDR < 0.1) in Molm13 PPM1D-mutant/cntrl for either the baseline or the AraC-treated comparisons and at the same time upregulated (FDR < 0.1) after PPM1D inhibitor GSK2830371 treatment. The combined occurrence of glutamine at +1 and ≥2 acidic residues in the flanking region is statistically significant (P = 3.595e-13, Fisher exact test). (F) Schema illustrating the components of the DDR pathway that are targeted by PPM1D in leukemia cells, based on the results from the phosphoproteomic analysis. Phosphorylation targets of PPM1D identified by mass spectrometry (FDR < 0.1) are shown in green. Predicted PPM1D target sites that were based on the identified consensus sequence with a glutamine at +1 and ≥2 acidic residues are shown in blue.

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