Journal:

Article Title: Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation

doi: 10.1093/embo-reports/kvd090

Figure Lengend Snippet: Fig. 2. Calcineurin is not involved in the inhibition of JNK and p38 pathways. (A) PKA-phosphorylated [32P]RII protein was incubated with 200 nM calcineurin in the presence of 500 nM calmodulin with or without 1 µM FR901725. Released 32P was measured as described in Methods. (B) Jurkat cells were pre-treated with the indicated concentrations of FR901725 for 1 h, followed by stimulation with TPA and A23187. The cells were then assayed for phosphorylation status of NFAT1 as well as JNK activity. This experiment is a representative of two. (C) Jurkat cells were transfected with pNFAT-Luc (left panel) or HA-JNK (right panel) along with the indicated amount of Cabin1, RHR4 and A238L expression vectors, followed by incubation for 40 h. The cells were stimulated with TPA and A23187 (T+I) in the presence (+FK) or absence of FK506 for 8 h and assayed for luciferase activities (left panel), or stimulated for 15 min and then assayed for JNK activity (right panel). This experiment is a representative of three. (D) Schematic diagram showing the effects of various inhibitors on the calcineurin–NFAT pathway and the JNK and p38 activation pathways.

Article Snippet: GST-fused RII peptide ( Blumenthal et al. , 1986 ), referred to here as RII protein, immobilized on glutathione–Sepharose was phosphorylated by PKA catalytic subunit (Sigma) in the presence of [γ- 32 P]ATP.

Techniques: Inhibition, Incubation, Activity Assay, Transfection, Expressing, Luciferase, Activation Assay

Journal:

Article Title: Molecular Determinants for PP2A Substrate Specificity: Charged Residues Mediate Dephosphorylation of Tyrosine Hydroxylase by the PP2A/B? Regulatory Subunit

doi: 10.1021/bi902160t

Figure Lengend Snippet: B′β Glu153 recognizes selectivity determinants close to the TH Ser40 phosphorylation site. (A) Diagram of glutathione S-transferase (GST) TH fusion proteins used in this figure. (B–F) Wild-type and mutant B′β-containing PP2A holoenzymes were isolated via FLAG immunoprecipitation (IP) from stable PC12 cell lines (B, C, E, and F) or from transiently transfected COS-1 cells (D). Enzyme aliquots containing equivalent C subunit (insets of panels B, D, and E) were assayed toward the indicated 32P-labeled GST fusion proteins (in vitro phosphorylated by PKA on TH Ser40 and Mfn2 Ser442). Representative phosphatase assays are shown in panels B, D, and E (mean ± standard deviation of triplicate reactions), while data summaries are shown in panels C and F (mean ± SE of three assays). An asterisk indicates p< 0.05.

Article Snippet: Preparation of Phosphatase Substrates GST–TH fusion protein, GST–Mfn2 fusion protein, and myelin basic protein (Sigma, St. Louis, MO) (3 μg/μL) were phosphorylated with the bovine heart PKA catalytic subunit [0.5 unit/μL (Sigma)] or recombinant ERK2 [5 ng/μL (Millipore/Upstate Biotech)] for 1 h at 30 °C in buffer containing 300 μM ATP, 50 μCi of [γ- 32 P]ATP, 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM dithiothreitol, 1 mM EGTA, and 0.01% Triton X-100.

Techniques: Mutagenesis, Isolation, Immunoprecipitation, Transfection, Labeling, In Vitro, Standard Deviation

Journal:

Article Title: Molecular Determinants for PP2A Substrate Specificity: Charged Residues Mediate Dephosphorylation of Tyrosine Hydroxylase by the PP2A/B? Regulatory Subunit

doi: 10.1021/bi902160t

Figure Lengend Snippet: Truncation of Mfn2 to the PKA phosphorylation site renders dephosphorylation dependent on Glu153 of B′β. (A) Diagram of GST–Mfn2 fusion proteins used in this figure and alignment of the PKA phosphorylation sites (arrow) in Mfn2 and TH. (B and C) PKA-phosphorylated Mfn2 fusion proteins were assayed for dephosphorylation by wild-type or mutant PP2A/B′β. A representative assay is shown in panel B (mean ± standard deviation of triplicate experiments) and a summary in panel C (ratio of wild-type B′β and E153R B′β activities; mean ± SE of three to seven assays). An asterisk indicates p< 0.05.

Article Snippet: Preparation of Phosphatase Substrates GST–TH fusion protein, GST–Mfn2 fusion protein, and myelin basic protein (Sigma, St. Louis, MO) (3 μg/μL) were phosphorylated with the bovine heart PKA catalytic subunit [0.5 unit/μL (Sigma)] or recombinant ERK2 [5 ng/μL (Millipore/Upstate Biotech)] for 1 h at 30 °C in buffer containing 300 μM ATP, 50 μCi of [γ- 32 P]ATP, 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM dithiothreitol, 1 mM EGTA, and 0.01% Triton X-100.

Techniques: De-Phosphorylation Assay, Mutagenesis, Standard Deviation

Journal:

Article Title: Molecular Determinants for PP2A Substrate Specificity: Charged Residues Mediate Dephosphorylation of Tyrosine Hydroxylase by the PP2A/B? Regulatory Subunit

doi: 10.1021/bi902160t

Figure Lengend Snippet: Arg37 and Arg38 are required for efficient dephosphorylation of TH by wild-type but not Glu153 mutant PP2A/B′β. (A) Sequence of the wild-type and Arg37 and Arg38 mutant TH–GST fusion protein and peptide, indicating ERK (Ser31) and PKA (Ser40) phosphorylation sites. (B and C) The indicated proteins were phosphorylated in vitro with ERK2 and assayed for dephosphorylation by wild-type and mutant PP2A/B′β: (B) representative assay showing the mean ± standard deviation of triplicate experiments and (C) summary of specific activities normalized to reactions with wild-type proteins (mean ± SE of three to five assays). (D) Phospho-Ser40 containing wild-type and Arg mutant TH peptides were assayed for dephosphorylation by wild-type and mutant PP2A/B′β using a colorimetric assay. Phosphatase activities are shown as means ± SE of three to five independent assays normalized to wild-type TH peptide. An asterisk indicates p< 0.05.

Article Snippet: Preparation of Phosphatase Substrates GST–TH fusion protein, GST–Mfn2 fusion protein, and myelin basic protein (Sigma, St. Louis, MO) (3 μg/μL) were phosphorylated with the bovine heart PKA catalytic subunit [0.5 unit/μL (Sigma)] or recombinant ERK2 [5 ng/μL (Millipore/Upstate Biotech)] for 1 h at 30 °C in buffer containing 300 μM ATP, 50 μCi of [γ- 32 P]ATP, 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 2 mM dithiothreitol, 1 mM EGTA, and 0.01% Triton X-100.

Techniques: De-Phosphorylation Assay, Mutagenesis, Sequencing, In Vitro, Standard Deviation, Colorimetric Assay