Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: Ey a3 directly interacts with Myc. A , Coomassie stain of FLAG-Eya3 overexpressed and purified from E. coli . B , Coomassie stain of His-Myc 1-88 overexpressed and purified from E. coli . C , purified FLAG-Eya3 and His-Myc 1-88 were incubated, immunoprecipitated using an α-FLAG antibody, followed by probing of His-Myc 1-88 with an α-Myc antibody in Western blot. The amount of FLAG-Eya3 used in immunoprecipitation was shown with Coomassie stain under the Western Blot. D , co-IP of FLAG-Eya3 CTD (residues 241–526) from HEK cells, followed by α-FLAG and α-Myc Western blot showed that the Eya3 CTD interacts with endogenous Myc. E , co-IP using an α-B55α antibody probed with α-B55α and α-Myc antibodies in 66cl4 cells with shRNA scramble control and α-Eya3 shRNA showed that Eya3 KD led to reduced association between B55α and Myc. F , quantification of data from panel E . G , co-IP using an α-B55α antibody probed with α-B55α and α-Myc antibodies in 66cl4 cells with and without FLAG-Eya3 overexpression (OE) showed that Eya3 OE led to increased association between B55α and Myc. H , quantification of data from panel G . ∗ p < 0 . 05 , ∗∗∗ p < 0.001 from four biological replicates in panel F and two biological replicates in panel H . Statistical analysis was done using an Fmax test followed by a two-tailed, homoscedastic t test. Error bars represent SD. CTD, C-terminal domain; Eya, eyes absent; IP, immunoprecipitation; KD, knockdown.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Staining, Purification, Incubation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, shRNA, Control, Over Expression, Two Tailed Test, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: Eya3 increases the phosphatase activity of PP2A-B55α but has no effect on the phosphatase activity of PP2A-B56α. A , PP2A-B55α phosphatase activity with the KRpTIRR peptide substrate in the presence and absence of FLAG-mouse Eya3 (mEya3) purified from E. coli . B , PP2A-B55α phosphatase activity with KRpSIRR substrate in the presence and absence of FLAG-mEya3. C , PP2A-B56α phosphatase activity with the KRpTIRR substrate in the presence and absence of FLAG-mEya3. D , PP2A-B56α phosphatase activity with the KRpSIRR substrate in the presence and absence of FLAG-mEya3. All phosphatase activities were determined using a malachite green assay with absorbance measured at 620 nm ( A 620 ). All statistical analysis was done on three biological replicates using an Fmax test followed by a two-tailed, homoscedastic t test. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Error bars represent the SD. Eya, eyes absent; PP2A, protein phosphatase 2A.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Activity Assay, Purification, Malachite Green Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: Eya3 uses a short, intrinsically disordered segment within its NTD to interact with B55α. A , AlphaFold prediction of Eya3. Very high confidence (predicted local distance difference test (pLDDT) > 90) was blue , high confidence (90 > pLDDT > 70) was light blue , low confidence (70 > pLDDT > 50) was yellow , and very low confidence (pLDDT <50) was orange . Residues (53–90) shown to bind B55α in panels C – E were shown in green . B , circular dichroism of purified Eya3 NTD (residues 1–287) confirmed it was disordered. C – E , FLAG-Eya3 with serial deletions within residues 53 to 120 and HA-B55α were cotransfected into HEK cells. FLAG co-IP followed by α-FLAG and α-HA probing in Western blot showed residues 53 to 90 were important for interacting with B55α. The ratio of HA-B55α to Flag-Eya3 in co-IPs were quantified in panel C with the ratio of WT B55a to Flag-Eya3 normalized to 1. p values were calculated from three biological replicates using a single-factor ANOVA, followed by Tukey’s test and the Fmax test, then a homoscedastic t test with the Bonferroni correction. ∗ p < 0.05, ∗∗∗ p < 0.001. Error bars represented the SD. Eya, eyes absent; IP, immpunoprecipitation; NTD, N-terminal domain.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Circular Dichroism, Purification, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: B55α interacts with Eya3 differently than its interactions with substrates. A – C , FLAG-Eya3 and HA-B55α carrying various mutations were cotransfected into HEK cells. FLAG co-IP was performed followed by α-FLAG and α-HA probing in Western blot. Lanes 10 and 11 were transfected with Eya3 or B55α alone as negative controls. Lane 1 was transfected with both WT Eya3 and B55α as a positive control, and lane 9 was transfected with WT B55α and Eya3 H79A mutant which is known to disrupt its interaction with B55α . The ratio of HA-B55α to Flag-Eya3 in co-IPs were quantified in panel C with the ratio of WT B55a to Flag-Eya3 normalized to 1. p values were calculated from three biological replicates using a single-factor ANOVA, followed by Tukey’s test and the Fmax test, then a homoscedastic t test with the Bonferroni correction. ∗ p < 0.05, ∗∗ p < 0.01. Error bars represented the SD. D , the B55α structure (gray, PDB ID: 3DW8 ) with residues that interacted with pTau in sticks for single amino acids and in colored ribbon for a range of amino acids (such as residues 84–90). Teal indicated residues that interact with both pTau and Eya3. Red indicated residues that interacted with pTau but did not interact with Eya3. Eya, eyes absent.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Positive Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: Eya3 and B55α KD affect the same phosphosite motifs. HEK cells with Eya3 or B55a KD were subjected to phosphoproteomic analyses. A , number of phosphosites significantly affected (log2FC >0.585, FDR <0.05 from three biological replicates for each KD) by Eya3 or B55α KD. B , phosphoserine (pS) and phosphothreonine (pT) amino acid motifs found enriched in WT compared to Eya3 KD cells. C , pS and pT amino acid motifs found enriched in WT compared to B55α KD cells. The dataset comprised two distinct biological replicates in panels B and C . Each dot (.) in the motif indicated an amino acid of any identity. Eya, eyes absent; FDR, false discovery rate; KD, knockdown.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Phospho-proteomics, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Biochemical characterization of the Eya and PP2A-B55α interaction

doi: 10.1016/j.jbc.2024.107408

Figure Lengend Snippet: PP2A-B55α prefers a non-Pro residue following phospho-Thr or Ser. A , malachite green assay showing that PP2A-B55α preferred Pro before pThr, not after pThr, on the generic substrate KRpTIRR. Residues different from the parental peptide are underlined in panels A and C – E . B , PP2A-B55α had much higher activity when Pro was mutated to Ala following pThr or pSer in the Myc peptide. Eya3 increased PP2A-B55α activity but did not change this preference. C , PP2A-B55α phosphatase activity with the DDpTPRG Ctps1 and DDpTARG mutant peptide substrates. D , PP2A-B55α phosphatase activity with the SSSpSPDS Dock7 and SSSpSADS mutant peptide substrates. E , PP2A-B55α phosphatase activity with the NPpSPPPD Eya1 and NPpSAPPD mutant peptide substrates. Absorbances were measured at 620 nm ( A 620 ) after incubating for 15 min in panel A or 30 min in panels B – E . Statistical analysis was done on three biological replicates using Fmax tests followed by two-tailed, homoscedastic t tests. In this figure, ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05. Error bars represented the SD. Eya, eyes absent; PP2A, protein phosphatase 2A.

Article Snippet: The following primary antibodies were used: α-FLAG (mouse, Sigma F3165), α-HA (rat, Roche 3F10), α-B55α (mouse, Santa Cruz sc-81606), α-EYA3 (rabbit, Proteintech 21196-1-AP), α-Myc pT58 (rabbit, ABM Y011034), α-β-actin (mouse, Santa Cruz sc-47778), α-Myc (rabbit, Abcam Y69), and α-GAPDH (mouse, GeneTex GT239).

Techniques: Residue, Malachite Green Assay, Activity Assay, Mutagenesis, Two Tailed Test