Fig. 3: The lack of fortilin promotes the phosphorylation, ubiquitination, and proteasomal degradation of CTNNA3.

FLAG-CTNNA3^WT (also WT) a mammalian plasmid containing the construct for FLAG-tagged wild-type human CTNNA3 (pEZ-CTNNA3^WT-FLAG), Pro-Q Diamond phosphoprotein gel staining, SYPRO Ruby total protein gel staining, CTNNA3^5D (also 5D) CTNNA3 protein with the following phosphomimetic mutations—S637D, S647D, T649D, S650D, and T653D; CTNNA3^5A (also 5A), CTNNA3 protein with the following phospho-null mutations—S637A, S647A, T649A, S650A, and T653A, THP1^WT-fortilin THP1 cells expressing wild-type fortilin, THP1^KO-fortilin THP1 cells in which the fortilin gene was deleted using Crispr-Cas9 technology, IP immunoprecipitation, α-FLAG anti-FLAG antibody (Ab), α-His₆, anti-His₆ Ab, JESS™ an automated capillary-based quantitative western blot system, Ni-NTA pulldown using Ni-NTA Superflow beads, A.U. arbitrary unit, Ub_n-CTNNA3 polyubiquitinated CTNNA3, α-fortilin anti-fortilin Ab, TCE 2,2,2-trichloroethanol. a Top panel: Pro-Q Diamond staining of phosphorylated CTNNA3^WT in the presence (THP1^WT-fortilin) or absence (THP1^KO-fortilin) of fortilin in the cell. THP1 cells were transfected with the pEZ-CTNNA3^WT-FLAG plasmid. CTNNA3^WT-FLAG in the total cell lysates was immunoprecipitated by anti-FLAG Ab and subjected to SDS-PAGE. Phosphorylated CTNNA3^WT-FLAG was visualized by Pro-Q Diamond staining. Bottom panel: SYPRO Ruby staining of total CTNNA3^WT in the presence (THP1^WT-fortilin) or absence (THP1^KO-fortilin) of fortilin in the cell. The same eluates from the above experiment were subjected to SDS-PAGE. Total CTNNA3^WT-FLAG was visualized by SYPRO Ruby staining. b Wild-type, phosphomimetic (5D), and phospho-null (5A) CTNNA3 mutant constructs. c Top panel: Pro-Q Diamond staining of phosphorylated CTNNA3^WT and CTNNA3^5A proteins in the presence (THP1^WT-fortilin) or absence (THP1^KO-fortilin) of fortilin in the cell. THP1 cells were transfected by the pEZ-CTNNA3^WT-FLAG plasmid. Bottom panel: SYPRO Ruby staining of total CTNNA3^WT and CTNNA3^5A proteins in the presence (THP1^WT-fortilin) or absence (THP1^KO-fortilin) of fortilin in the cell. The same eluates from the above experiment were subjected to SDS-PAGE. Total CTNNA3^WT-FLAG and CTNNA3^5A proteins were visualized by SYPRO Ruby staining. d Impact of CTNNA3 phosphorylation on the degree of ubiquitination in 293T cells. 293T cells were transfected by the pEZ-CTNNA3^WT-FLAG, pEZ-CTNNA3^5D-FLAG, or pEZ-CTNNA3^5A-FLAG plasmid. Ubiquitinated wild-type and mutant CTNNA3s were visualized by first pulling down the ubiquitinated proteins using Ni-NTA beads and then immunoblotting CTNNA3 within the pulled-down proteins using α-FLAG Ab in the JESS™ system. e The CTNNA3 ubiquitination indices of wild-type and mutant CTNNA3s. The indices (in A.U.) were calculated by dividing the area under the curve of the ubiquitinated CTNNA3-FLAG by that of the input CTNNA3-FLAG. Data are expressed as means ± s.d. (n = 3 biological replicates), and they were analyzed by one-way ANOVA and Tukey–Kramer multiple-comparisons. f, g Impact of CTNNA3 phosphorylation on its proteasome-mediated degradation. 293T cells were transfected by the pEZ-CTNNA3^WT-FLAG, pEZ-CTNNA3^5D-FLAG, or pEZ-CTNNA3^5A-FLAG plasmid and incubated in the presence of CHX and with or without MG132 for various durations. CTNNA3-FLAGs were detected by anti-FLAG Ab, and total proteins were visualized using the JESS™ total protein detection module. CTNNA3 expression indices (in A.U.) were calculated using Compass software by dividing the area under the curve of a CTNNA3 peak by the total proteins loaded in the same capillary (“in-capillary normalization”). Data are expressed as means ± s.d. (n = 3 biological replicates). Statistical analyses were performed using one-way ANOVA with Fisher’s pairwise comparisons. h, i Co-IP analysis of the strength of the interaction between fortilin and wild-type and mutant CTNNA3s. The total cell lysates from 293T cells transfected by the pEZ-CTNNA3^WT-FLAG, pEZ-CTNNA3^5D-FLAG, or pEZ-CTNNA3^5A-FLAG plasmid were subjected to IP by α-FLAG Ab. Co-immunoprecipitated fortilin was then detected by immunoblotting using α-fortilin Ab. The fortilin-CTNNA3 interaction indices (in A.U.) were calculated using ImageJ software by dividing the signal intensities of co-immunoprecipitated fortilin bands by the immunoprecipitated CTNNA3-FLAG bands. Data are expressed as means ± s.d., (n = 3 biological replicates), and they were analyzed by one-way ANOVA and Tukey–Kramer tests.