Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 5. Purified Xenopus PP2C dephosphorylates the Thr-160 residue of Cdk2. A, Xenopus PP2C was purified according the proce- dure described in Table I. The active fractions of various purification steps (P60, DEAE-Sepharose, Phenyl-Superose: PS; Hi-Trap Blue: Blue) were pooled, electrophoresed, and Western-blotted with the anti- PP2C antibody (left panel). Proteins from the active fraction of the last purification step (Hi-Trap Blue column) were visualized by Amido Black staining (right panel). MWM, molecular weight markers. B, re- combinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP, then incubated for 20 min in the presence of 0.1 g of the active fraction of the last purification step (Hi-Trap Blue column) and increasing concentrations of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Purification, Residue, Western Blot, Staining, Molecular Weight, Incubation

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 6. Xenopus recombinant PP2C dephosphorylates the Thr- 160 residue of Cdk2. A, recombinant Cdk2 and / casein were first phosphorylated in the presence of [-32P]ATP by Cak1 and PKA, re- spectively. Phosphorylated Cdk2 or casein were then incubated for 20 min in the presence of 2 g Xenopus recombinant PP2C protein, in the presence or in the absence of Mg2 and 106 M okadaic acid, and the percentage of dephosphorylated substrate was measured. B, recombi- nant Cdk2 (5 M) was first phosphorylated by Cak1 in the presence of cold ATP and then incubated for 30 min at 30 °C with increasing concentrations (1.2, 0.24, 0.12, 0.024, and 0.012 mg/ml) of Xenopus recombinant PP2C. Proteins were Western-blotted with an antibody recognizing specifically the phospho-Thr-160 residue of Cdk2 (lower panel) and an anti-PSTAIR antibody recognizing all forms of Cdk2 (upper panel). C, recombinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP. Phosphorylated Cdk2 was incubated for 30 min in the presence of increasing concentrations of recombinant cyclin A. Xenopus recombinant PP2C (2 g) was then added in the presence of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Recombinant, Residue, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 7. Monomeric Cdc2 in P60 and F9 is phosphorylated on Thr-161. A, P40 and P60 were prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). They were analyzed by Western blotting with the anti-cyclin B2 antibody (upper panel), the anti-Cdc2 antibody (middle panel), and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). B, F9 was prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). As indicated, 25 mM Mg2 with or without Cak1 was added back or not in F9 initially prepared in the absence of Mg2, and the fraction was further incubated for 30 min at 30 °C before Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). C, F9 was prepared in the presence of Mg2 and then incubated in the presence or in the absence of Cak1 for 30 min at 30 °C. PP2C (0.9 mg/ml in the final assay) was added or not, and then incubation was extended for 30 min. Samples were analyzed by Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho-Thr-161 residue of Cdc2 (lower panel).

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Western Blot, Residue, Incubation

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 6. Xenopus recombinant PP2C dephosphorylates the Thr- 160 residue of Cdk2. A, recombinant Cdk2 and / casein were first phosphorylated in the presence of [-32P]ATP by Cak1 and PKA, re- spectively. Phosphorylated Cdk2 or casein were then incubated for 20 min in the presence of 2 g Xenopus recombinant PP2C protein, in the presence or in the absence of Mg2 and 106 M okadaic acid, and the percentage of dephosphorylated substrate was measured. B, recombi- nant Cdk2 (5 M) was first phosphorylated by Cak1 in the presence of cold ATP and then incubated for 30 min at 30 °C with increasing concentrations (1.2, 0.24, 0.12, 0.024, and 0.012 mg/ml) of Xenopus recombinant PP2C. Proteins were Western-blotted with an antibody recognizing specifically the phospho-Thr-160 residue of Cdk2 (lower panel) and an anti-PSTAIR antibody recognizing all forms of Cdk2 (upper panel). C, recombinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP. Phosphorylated Cdk2 was incubated for 30 min in the presence of increasing concentrations of recombinant cyclin A. Xenopus recombinant PP2C (2 g) was then added in the presence of Mg2, and the release of 32P from Cdk2 was measured.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Recombinant, Residue, Incubation, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Phosphodiesterase 4 is overexpressed in keloid epidermal scars and its inhibition reduces keratinocyte fibrotic alterations.

doi: 10.1186/s10020-024-00906-8

Figure Lengend Snippet: Fig. 6 Mechanistic studies (I): Roflumilast prevents TGFβ1-induced phospho-SMAD3 by the increase of PPM1A, the inhibition of reactive oxygen species (ROS) and phospho-ERK1/2-Protein Kinase A axis. Human keratinocytes were incubated for 30 min with vehicle or roflumilast (Rof, 100 nM) (A-F), the SMAD3 inhibitor (SIS3, 10µM) (A), the anti-oxidant N-acetyl-L-cysteine (NAC, 1 mM) (B, E), the PPM1A inhibitor sanguinarine (San, 3 µM) (D), the protea some inhibitor MG132 (MG132, 5 µM) (E), the PKA inhibitor (KT5720, 2µM) (C), the ERK1/2 inhibitor (PD98059, 10µM) (F) or combinations as indicated. Next, keratinocytes were stimulated with TGFβ1 (10 ng/ml) for 30 min and p-SMAD3 and PPM1A proteins were analysed by Western blotting. Data are shown from densitometry as the ratio of target protein compared to β-actin of three independent experiments per condition. Results of the densitometry are presented as scatter dot blot with median and interquartile range values. P-values are based on Kruskal-Wallis test followed by the Dunn’s post-hoc test

Article Snippet: The membrane was blocked with 5% Marvel in PBS containing 0.1% Tween20 (PBS-T), probed with the following antibodies: PDE4B2 (Sigma-Aldrich, cat no ABS181), phospho-SMAD3 (Novus Biologicals, cat.no NBP1-77836; RRID: AB_11031542), phospho-ERK1/2 (Sigma Aldrich, cat.no M9692), αSMA (Sigma-Aldrich, cat. no A5228), collagen type I (Novus Bilogicals, cat.no NB600-408; RRID: AB_10000511), PPM1A (Cell signaling, cat. no 3549 S; RRID: AB_2169764), phospho-PDE4B/D ERK site antibody (FabGennix, cat, no PPD4-450AP; RRID: AB_3095755), E-cadherin (ECM Bioscience, cat, no CM1681; RRID: AB_2076812), NOX4 (Novus, cat. no NB110-58849; RRID: AB_877739) and normalized to total anti-human/mouse β-actin (1:1000) antibody (42 KDa, monoclonal antibody, catalog no. A1978; RRID: AB_476692, Sigma Aldrich).

Techniques: Inhibition, Incubation, Western Blot, Dot Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Phosphodiesterase 4 is overexpressed in keloid epidermal scars and its inhibition reduces keratinocyte fibrotic alterations.

doi: 10.1186/s10020-024-00906-8

Figure Lengend Snippet: Fig. 7 Mechanistic studies (II): Roflumilast prevents TGFβ1-induced phospho-ERK1/2 by the reduction of reactive oxygen species (ROS), the increase of protein kinase A (PKA) and PPM1A. Human keratinocytes were incubated for 30 min with vehicle or roflumilast (Rof, 100 nM) (A-F), the ERK1/2 inhibitor (PD98059, 10µM) (A), the anti-oxidant N-acetyl-L-cysteine (NAC, 1 mM) (B), the PPM1A inhibitor sanguinarine (San, 3 µM) (D), the PKA inhibitor (KT5720, 2µM) (C), the SMAD3 inhibitor (SIS3, 10µM) (F) or combinations as indicated. Next, keratinocytes were stimulated with TGFβ1 (10 ng/ml) for 30 min and p- SMAD3 and PDE4D/B proteins were analysed by Western blotting. Data are shown from densitometry as the ratio of target protein compared to β-actin of three independent experiments per condition. Results of the densitometry are presented as scatter dot blot with median and interquartile range values. P-values are based on Kruskal-Wallis test followed by the Dunn’s post-hoc test

Article Snippet: The membrane was blocked with 5% Marvel in PBS containing 0.1% Tween20 (PBS-T), probed with the following antibodies: PDE4B2 (Sigma-Aldrich, cat no ABS181), phospho-SMAD3 (Novus Biologicals, cat.no NBP1-77836; RRID: AB_11031542), phospho-ERK1/2 (Sigma Aldrich, cat.no M9692), αSMA (Sigma-Aldrich, cat. no A5228), collagen type I (Novus Bilogicals, cat.no NB600-408; RRID: AB_10000511), PPM1A (Cell signaling, cat. no 3549 S; RRID: AB_2169764), phospho-PDE4B/D ERK site antibody (FabGennix, cat, no PPD4-450AP; RRID: AB_3095755), E-cadherin (ECM Bioscience, cat, no CM1681; RRID: AB_2076812), NOX4 (Novus, cat. no NB110-58849; RRID: AB_877739) and normalized to total anti-human/mouse β-actin (1:1000) antibody (42 KDa, monoclonal antibody, catalog no. A1978; RRID: AB_476692, Sigma Aldrich).

Techniques: Incubation, Western Blot, Dot Blot

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 1. Separation of monomeric Cdc2 from cyclin BCdc2 het- erodimer. A, the 100,000 g (S100) cytosolic extract and the P40 and the P60 ammonium precipitates were analyzed by Western blotting, using (from the top panel to the lower panel) anti-cyclin B2 antibody, anti-cyclin B1 antibody, anti-Cdc2 antibody, anti-phospho-Tyr-Cdc2 an- tibody (P-Tyr Cdc2). B, Superose 12 chromatography of P40 and P60. Optical density (OD) was measured at 280 nm in the Superose 12 fractions. Molecular mass markers (Amersham Biosciences) are indi- cated. The Superose 12 fractions were analyzed by Western blotting with the antibodies against cyclin B2, Cdc2, and phospho-Tyr-Cdc2 (P-Tyr Cdc2). The fractions containing monomeric Cdc2 are indicated by the bottom arrows.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Western Blot, Chromatography

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 2. In vitro activation of Cdc2 by Cak1 and cyclins. A, activation of Xenopus recombinant wild type Cdc2 (wt) or T161A mu- tant Cdc2 (T161A). The histone H1 activity of recombinant proteins (1.5 g) was measured in vitro after incubation in the presence or not of recombinant Cak1 (0.2 g) and either His-cyclin B1 or GST-cyclin A (3 g). Upper panel, quantification of Cdc2 kinase activity, expressed in cpm incorporated in histone H1. Lower panel, autoradiogram of phos- phorylated histone H1. B, the 100,000 g (S100) cytosolic extract and the P40 and the P60 ammonium precipitates were analyzed by Western blotting with the anti-MO15/CDK7 antibody. C, activation of mono- meric Cdc2 in P60. The histone H1 kinase activity of endogenous Cdc2 in P60 (120 g of protein/assay, equivalent to 10 oocytes/assay) was measured in vitro after incubation in the absence or in the presence of Cak1 (0.2 g) and increasing concentrations of GST-cyclin A or His- cyclin B1. Cdc2 activity is expressed as a percentage of its maximum activity.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: In Vitro, Activation Assay, Recombinant, Activity Assay, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 3. A phosphatase present in P60 and F9 counteracts Cdc2 and Cdk2 activation by Cak1. A, increasing amounts of P60 were incubated with Cak1 (0.2 g) and GST-cyclin A (3 g or 1.5 M). The Cdc2cyclin A complexes were recovered on GSH beads, and the histone H1 kinase activity of Cdc2 was then measured. B, recombinant Cdk2 was first phosphorylated by Cak1 in the presence of [-32P]ATP. The phosphorylated protein was then incubated for 30 min with increasing amounts of P60 or F9. Phosphorylation of Cdk2 was detected by elec- trophoresis and autoradiography. The radioactivity incorporated in Cdk2 band was quantified and expressed as a percentage of the maximum.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Activation Assay, Incubation, Activity Assay, Recombinant, Phospho-proteomics, Autoradiography, Radioactivity

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 7. Monomeric Cdc2 in P60 and F9 is phosphorylated on Thr-161. A, P40 and P60 were prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). They were analyzed by Western blotting with the anti-cyclin B2 antibody (upper panel), the anti-Cdc2 antibody (middle panel), and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). B, F9 was prepared in the absence (Mg2) or in the presence of Mg2 (Mg2). As indicated, 25 mM Mg2 with or without Cak1 was added back or not in F9 initially prepared in the absence of Mg2, and the fraction was further incubated for 30 min at 30 °C before Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho- Thr-161 residue of Cdc2 (lower panel). C, F9 was prepared in the presence of Mg2 and then incubated in the presence or in the absence of Cak1 for 30 min at 30 °C. PP2C (0.9 mg/ml in the final assay) was added or not, and then incubation was extended for 30 min. Samples were analyzed by Western blotting with the anti-Cdc2 antibody (upper panel) and the antibody recognizing specifically the phospho-Thr-161 residue of Cdc2 (lower panel).

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Western Blot, Residue, Incubation

Journal: Journal of Biological Chemistry

Article Title: Thr-161 Phosphorylation of Monomeric Cdc2

doi: 10.1074/jbc.m202742200

Figure Lengend Snippet: FIG. 8. The subpopulation of monomeric Thr-161-phosphoryl- ated Cdc2 is activable by cyclin. P60 was prepared in the absence (Mg2) or in the presence of Mg2 (Mg2) and incubated in the presence or in the absence of GST-cyclin A (3 g) and Cak1 (0.2 g) in modified EB buffer (EDTA). As indicated, Mg2 was added back or not in P60 initially prepared in the absence of Mg2. Cdc2cyclin A com- plexes were recovered by GST pull-down. The pull-down samples were either assayed for histone H1 kinase activity (top panel) or Western- blotted with the following antibodies: anti-Cak1, anti-phospho-Thr-161 Cdc2, and anti-Cdc2 antibodies.

Article Snippet: Polyclonal rabbit anti-phospho-Cdc2 (Tyr-15) and anti-phospho-Cdc2 (Thr-161) antibodies were purchased from Cell Signaling Technology, and polyclonal rabbit anti-PSTAIR antibody and polyclonal sheep antihuman PP2C were purchased from Upstate Biotechnology.

Techniques: Incubation, Modification, Activity Assay, Western Blot