Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: Expression of constitutively active Src or Fyn kinase induces tyrosine phosphorylation of endogenous PP2Ac and HA-tagged WT and Y307F PP2Ac. A , lysates from control (−) and Src CA -transformed (+) MEFs were subjected to microcystin-Sepharose pull downs (MPDs). Total cell lysates and MPDs were analyzed by Western with antibodies recognizing phosphotyrosine (pY) and endogenous PP2Ac. Representative Western blots of total cell lysates and HA-immunoprecipitates prepared from SYF ( B ), N2a ( C ), or Cos-7 ( D and E ) cells transfected with either empty vector (EV), HA-/HA3-tagged wildtype PP2Ac (WT), or the Y307F phosphomutant of PP2Ac (Y307F) together with plasmids encoding Src CA , GFP-tagged Fyn CA , or EV. Where indicated, a subset of these cells was treated with pervanadate (+PV) prior to harvesting. Well-characterized anti-pY antibodies were used to assess tyrosine phosphorylation of proteins in response to SFK CA and pervanadate. Tubulin antibodies were used to control for protein loading. Similar results were observed in at least four separate experiments.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Expressing, Transformation Assay, Western Blot, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: Distinct effects of pervanadate, Scr CA , and Fyn CA on PP2Ac WT, Y127F, Y284F, and Y127/284F tyrosine phosphorylation. Lysates and HA-immunoprecipitates were prepared from control and pervanadate-treated (+PV) Cos-7 cells ( A and B ), Src CA -transfected Cos-7 cells ( C and D ), and GFP-Fyn CA -transfected N2a cells ( E ) that were cotransfected with plasmids encoding HA3-tagged PP2Ac wildtype (WT), Y127F (Y127F), Y284F (Y284F), Y127/284F (F/F), or the empty vector (EV). Representative Western blots with the indicated antibodies are shown in ( A ), ( C ), and ( E ). Note: the pSFK antibody recognizes Src only when phosphorylated at Y416. B , relative levels of pY signal in HA3-PP2Ac immunoprecipitates from ( A ). Data (mean ± SEM from n = 3 separate experiments) were appraised using one-way ANOVA (F = 236; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared with WT; #### p < 0.0001, # p < 0.05. D , relative levels of pY signal in HA3-PP2Ac immunoprecipitates from ( C ). Data (mean ± SEM from n = 3–4 separate experiments) were appraised using one-way ANOVA (F = 2067; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared with WT; #### p < 0.0001. ( E ) similar results were observed in three separate experiments.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: Western analyses using newly generated phosphosite-specific antibodies reveal that PP2Ac is differentially phosphorylated by Fyn CA and Src CA at the Y284 and Y127 epitopes. A , representative Western blots of HA-immunoprecipitates prepared from N2a cells coexpressing HA3-tagged PP2Ac and either GFP- Fyn CA or an empty vector (EV) and probed with mouse anti-HA, alpaca anti-pY284 (pY284), or rabbit anti-pY127 (pY127) PP2Ac antibodies. Where indicated, a subset of these cells were treated with pervanadate (+PV) prior to harvesting. B , Western analysis of HA-immunoprecipitates prepared from EV- or Src CA -transfected Cos-7 cells that were cotransfected with plasmids encoding HA3-tagged PP2Ac wildtype (WT), Y127F (Y127F), Y284F (Y284F), or Y127/284F (F/F). C and D , Western analyses of total cell lysates and FLAG-immunoprecipitates prepared from Cos-7 cells that were cotransfected with plasmids encoding Src CA , FLAG-tagged Bα regulatory subunit (FLAG-Bα), and/or the empty vector (-). Blots were probed with anti-PP2Ac, anti-PP2A B subunit, anti-pY ( C ), or the novel PP2Ac phosphospecific Y284 and Y127 ( D ) antibodies. Where indicated, a subset of these cells was treated with pervanadate (PV) prior to harvesting. E , Western analyses of HA-immunoprecipitates prepared from Cos-7 cells that were cotransfected with plasmids encoding Src CA and HA-tagged PP2Ac WT or the L309Δ, D88N, and H59Q mutants. Expression of Src CA in the lysates was verified using the pY416-SFK antibody.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Western Blot, Generated, Plasmid Preparation, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: SFK-mediated tyrosine phosphorylation of PP2Ac decreases its binding to the regulatory Bα subunit. A , total lysates and HA-immunoprecipitates were prepared from empty vector (EV)- or Src CA -transfected Cos-7 cells that were cotransfected with plasmids encoding either HA3-tagged PP2Ac wildtype (WT), Y127F (Y127F), Y284F (Y284F), or Y127/284F (F/F). Blots were analyzed using anti-Bα, anti-PP2Ac, or anti-HA antibodies. B , relative levels of Bα were quantified in HA3-PP2Ac immunoprecipitates. Data are mean ± SEM from at least n = 4 separate experiments and were appraised using unpaired t -tests as indicated. ∗ p < 0.05; ∗∗∗∗ p < 0.0001, Src CA versus EV; ns, not significant. C , Western blots of total lysates and HA-immunoprecipitates prepared from empty vector (EV)- or GFP-Fyn CA -transfected N2a cells that were cotransfected with plasmids encoding either HA3-tagged PP2Ac wildtype (WT), Y284F (Y284F), or Y127F (Y127F). Levels of GFP-Fyn and Bα in the input used for immunoprecipitation (lysates) are shown for reference. D , relative levels of Bα binding in HA3-PP2Ac immunoprecipitates. Data are mean ± SEM from n = 3 separate experiments and were appraised using one-way ANOVA (F = 29.02; p = 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Binding Assay, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: PP2Ac phosphomutants exhibit decreased interactions with SFK CA . A , duplicate HA3-PP2Ac immunoprecipitates from the same transfected Cos-7 cells analyzed in Figure 2 C were subjected to Western analysis using anti-pY416 SFK and anti-HA antibodies. B , relative levels of Src CA in HA3-PP2Ac immunoprecipitates. Data are mean ± SEM from n = 4 separate experiments and were appraised using one-way ANOVA (F= 97.17; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗ p < 0.05, ∗∗∗∗ p < 0.0001, compared with WT; # p < 0.05. C , representative Western blots of total lysates and HA-immunoprecipitates prepared from empty vector (EV)- or GFP-Fyn CA -expressing N2a cells that were cotransfected with plasmids encoding either HA3-tagged PP2Ac wildtype (WT), Y284F (Y284F), Y127F (Y127F), or Y127/284F (F/F). D , relative levels of Fyn CA in HA3-PP2Ac immunoprecipitates. Data are mean ± SEM from n = 3 separate experiments and were appraised using one-way ANOVA (F = 59.07; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared to WT; ns, not significant.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: The Y127/284F and Y284F phosphoincompetent mutants inhibit Src CA - and Fyn CA -mediated ERK activation, respectively. A , Cos-7 cells were transfected with plasmids encoding HA3-tagged PP2Ac WT, Y127F, Y284F, or the empty vector (EV). Total cell homogenates from serum-starved cells were analyzed by immunoblotting for active ERK using validated antibodies recognizing the phosphorylated forms of ERK2 (or p42-MAPK, lower band) and ERK1 (or p44-MAPK, upper band), and total ERK1/2. PP2Ac expression levels in the cells are shown as reference. Pervanadate-treated control cells and WT-transfected cells incubated with the SFK inhibitor, PP2, were used as controls. B , Western analyses of active ERK in Cos-7 cells that were cotransfected with plasmids encoding Src CA and HA3-tagged WT, Y127F, Y284F, F/F or Y307F PP2Ac, or the empty vector (EV). Extracts from EV-transfected cells served as controls. HA3-PP2Ac and Src expression levels in these cells are shown as reference. C , relative phosphorylated ERK (pERK) levels in Cos-7 cells transfected as in B . Data are mean ± SEM from at least three separate experiments and were appraised using one-way ANOVA (F = 45.13; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, compared to WT + Src CA ; # p < 0.05; ns, not significant. D , representative Western blots of cell homogenates prepared from control Src CA -, or Fyn CA -transfected N2a cells that were cotransfected with an empty vector (EV), HA3-tagged PP2Ac wildtype (WT), Y127F, Y284F, or F/F. Duplicate aliquots of the cell lysates were analyzed for active ERK, HA-PP2Ac, Src, and GFP-Fyn expression levels. Note that top ERK panels were assembled from the same blot. E , relative phosphorylated ERK (pERK) levels in N2a cells transfected as in D . Data are mean ± SEM from n = 3 separate experiments and were appraised using one-way ANOVA (F = 66.78; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared with WT + Src CA ; #### p < 0.0001; ns, not significant.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: PP2Ac phosphomutants inhibit Src CA - and Fyn CA -mediated Tau phosphorylation. A , the same total cell homogenates from control ( Fig. 6 D , left panel ), Src CA - ( Fig. 6 D , center panel ), or Fyn CA - ( Fig. 5 C , right panel ) transfected N2a cells that were cotransfected with EV or HA-PP2Ac species were reanalyzed by Western for endogenous Tau phosphorylation at the CP13 phosphoepitope (pSer202) and for total Tau. B , relative Ser 202-phosphorylated Tau (p-Tau) levels in N2a cells transfected as in A . Data are mean ± SEM from n = 3 separate experiments and were appraised using one-way ANOVA (F = 61.67; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗ p < 0.05, ∗∗∗∗ p < 0.0001, compared with WT + SFK CA ; ns, not significant. C , representative immunoblots of total and Ser 9-phosphorylated GSK-3β from the same cell lysates analyzed in A . Similar results were obtained in two other experiments.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Transfection, Western Blot