Fig 2: PPP1R12A is essential for the YAP-dependent proliferation of breast cancer cells. (a) Schematics of biotinylation of proteins proximal to PS with BirA*-2xPH (the PS biosensor). The list of phosphatases identified by the BioID method using BirA*-2xPH11 is shown. (b) MDA-MB-231 cells were treated with the indicated siRNA for 48 h and then replated. After 24 h, the cells were transfected with the same siRNA again and further incubated for 96 h. The cell number was counted, normalized to the mean value of control siRNA (siCtrl)-treated cells, and shown in a bar graph. (c) MDA-MB-231 cells were treated with the indicated siRNA, and the cell number was counted as in (b). (d–f) MDA-MD-231 cells were treated with the indicated siRNA as in (b), and the mRNA levels of PPP1R12A (d), LATS1 (e), and LATS2 (f) were determined by qRT-PCR and shown in a bar graph. GAPDH was used as an internal control. (g) MCF-7 cells were treated with the indicated siRNA as in (b), and the cell number was counted, normalized to the mean value of siCtrl-treated cells, and shown in a bar graph. (h) MCF-7 cells were treated with the indicated siRNA as in (b), and the mRNA level of PPP1R12A was determined by qRT-PCR. GAPDH was used as an internal control. Data represent the mean ± s.d. from duplicate or triplicate experiments with data points plotted. n.s., not significant; *p < 0.05; **p < 0.01 vs. siCtrl, one-way ANOVA with Tukey–Kramer post hoc test.

Fig 3: PCBP2 is essential for TRIB2 to regulate PSMB5 activity.a Venn diagram showing three biologically independent MS results from the Bel-7402 cell immunoprecipitation with anti-TRIB2 antibodies. The candidates were further screened by UniProt online software and the literature to identify potential ubiquitination-related proteins. b Representative Coomassie blue stain image of the Bel-7402 cell immunoprecipitates pulled down by anti-TRIB2 or IgG antibodies (n = 3). c Ub in the control and Bel-7402 cells with MYPT1, GRP78, PARP1, or PCBP2 knocked down. d Ub in the control and Bel-7402 cells overexpressing PCBP1, PCBP2, or PCBP3 with or without TRIB2 knocked down. e Reciprocal co-IP results of purified TRIB2 and PCBP2 proteins in vitro. TRIB1, TRIB3, PCBP1, and PCBP3 were parallel examined to exclude non-specificity. f Positive correlation between PCBP2 and TRIB2 in the liver cancer specimens. TMA was performed by IHC using anti-PCBP2 and anti-TRIB2 antibodies (n = 208). The data were analyzed by Spearman’s rank correlation. g The effects of TRIB2 on Ub level were PCBP2-dependent. Ub in the control and Bel-7402 cells with or without TRIB2 knocked out or overexpressed, in the presence or absence of PCBP2 knocking out. h PCBP2 regulated Ub via PSMB5. Ub in Bel-7402 cells with PCBP2 and PSMB5 knocked out or overexpressed, as indicated. i TRIB2 regulated PSMB5 activity via PCBP2. A proteasome activity assay kit (AAT Bioquest) was used to evaluate PSMB5 activity in the Bel-7402 and SMMC-7721 cells under the same treatment as indicated in g (n = 3). Data were analyzed by one-way ANOVA and expressed as mean ± SD. ****P < 0.0001; NS non-significance. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (c, d, g, h).

Fig 4: TIMAP reduces MYPT1 abundance in ECs. A, PP1cα and PP1cβ levels in ECs transduced with GFP-vector, GFP-TIMAPWT, or GFP-TIMAPV64A/F66A. Left panel, representative WB analysis. Right panel, densitometric analysis (mean ± S.D.; n = 5 independent experiments; *, p < 0.05 versus vector control; Student's t test). B, MYPT1 and pS696MYPT1 in ECs transduced with GFP-vector, GFP-TIMAPWT, or GFP-TIMAPV64A/F66A. Left panel, representative WB analysis. Right panel, densitometric analysis (mean ± S.D.; n = 5 independent experiments; *, p < 0.05 versus vector control; Student's t test). C, MYPT1 mRNA abundance determined by RT-PCR in ECs transduced with GFP-vector, GFP-TIMAPWT, or GFP-TIMAPV64A/F66A (mean ± S.D.; n = 5 independent experiments). D, effect of MG132 (10 μm) on MYPT1 and pMLC2 abundance in glomerular ECs transduced with GFP-vector or GFP-TIMAPWT. Left panel, representative WB analysis. Right panel, densitometric analysis (mean ± S.D., n = 4 independent experiments; *, p < 0.05, two-way ANOVA with post hoc Bonferroni analysis). E, MYPT1, PP1cβ, and pMLC2 abundance in ECs transduced with 2.5, 5, or 10 m.o.i. of GFP-vector, GFP-TIMAPS333A/S337A, or GFP-TIMAPS333E/S337E. Right panel, representative WB analysis. Left panel, densitometric analysis (values for 2.5 m.o.i. of vector were set at 1.0 (horizontal gray bar); individual data points from six to seven independent experiments are shown; *, p = 0.01; **, p < 0.001 versus GFP-vector control; two-way ANOVA with post hoc Bonferroni analysis).

Fig 5: Higher expression of ATP8A1, PPP1R12A, and PP1B correlates with shorter survival times in breast cancer patients. (a) Kaplan–Meier survival curves based on the expression levels of the indicated gene in cohorts of breast cancer (TCGA, n = 1091) were shown. The colored region along the curve showed the 95% confidence intervals. Number of patients in the each group were represented as ATP8A1 (High: n = 213, Low: n = 878), PP1A (High: n = 884, Low: n = 207), PP1B (High: n = 128, Low: n = 963), PP1C (High: n = 133, Low: n = 958), PPP1R8 (High: n = 197, Low: n = 894), PPP1R9A (High: n = 948, Low: n = 143), PPP1R10 (High: n = 800, Low: n = 291), PPP1R12A (High: n = 810, Low: n = 281), PPM1G (High: n = 982, Low: n = 109), PPP2R1A (High: n = 902, Low: n = 189), and PPP2R5D (High: n = 944, Low: n = 147). The expression data of PPP1R37 was not available in TCGA. Significant correlations between high expression levels and shorter survival periods were only observed for PPP1R12A and PP1B among the genes encoding the PS-proximal phosphatases. (b) A model of the regulation of YAP dephosphorylation on the RE membrane.