Fig 2: Aromatic and acidic residues flanking S67/S71 Gwl site are essential for slow Arpp19 dephosphorylation rate.A S67/S71 dephosphorylation of wild-type Arpp19 or of the indicated DSG Arpp19 mutants was measured in kinase-inactivated extracts depleted or not of B55, as well as upon the addition of a purified PP2A-B55 phosphatase by autoradiography (P33Arp). The amount of Arpp19 in each sample was assessed by western blot (Arp). The percentage of phosphorylation remaining with respect to the starting point calculated as in Fig. 1B and represented in bar graph as the mean value ± SD; n = 3 biological independent samples. B Dephosphorylation of the wild type and the indicated mutants of Arpp19 were performed in kinase-inactivated extracts and quantified in a bar graph as the mean value ± SD; n = 3 biological independent samples.

Fig 3: Putative docking of the DSG motif of Arpp19 into the active site of PP2A-B55.A The docking of phospho-S67/S71 of Arpp19 into PP2A catalytic subunit (C) as an inhibitor conformation, its rearrangement towards a substrate conformation and its final dissociation upon dephosphorylation. Phosphorylation of S67/S71 is represented (yellow circle). The two aspartic residues D70 and D73 (pink circles) that could interact with the metallic centre (red circles) and with the two arginine residues (R) of the PP2A C subunit are shown. The putative effects of KKR/AAA, G72A, and D70A/D73A mutants of Arpp19 on the docking conformation of phospho-S67/S71 are indicated. B Putative docking of the DSG motif of Arpp19 into the active site of PP2A-B55 (PDB2IE4)24. Left: conformation of the peptide orientating the phosphorylated serine (yellow circle) toward the catalytic centre with the two aspartates (red circles) pointing toward nearby arginines (R89 and R214). Right: inhibited form in which the phosphorylated serine points outside (toward R268), while the dications (violet spheres) from the catalytic centre are chelated by one or two aspartates from the DSG motif.

Fig 4: S67/S71 catalysis and PP2A-B55 interaction regulate each other to confer a proper timing of PP2A-B55 inhibition.A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t-tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t-tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E, except that the indicated mutant form of Arpp19 was used.

Fig 5: Phospho-S67/S71 negatively regulates dephosphorylation of S109/S113 of Arpp19 by PP2A-B55.A Arpp19 was phosphorylated ‘in vitro’ on S109/S113, S67/S71 or thio-phosphorylated on S67/S71, supplemented alone or with the indicated combinations to kinase-inactivated extracts and the temporal pattern of dephosphorylation analysed. B ENSA protein was phosphorylated ‘in vitro’ on S67 by GwlK72M or S109 by PKA and supplemented alone or combined as indicated to kinase-inactivated extracts and the dephosphorylation time of these residues, as well as the amount of ENSA protein examined. C Phospho-S109 ENSA and phospho-S67/S71 Arpp19 or phospho-S109/S113 Arpp19 and phospho-S67 ENSA were simultaneously added into kinase-inactivated extracts and the timing of dephosphorylation of the different phospho-sites analysed. Experiments supporting the data of this figure were performed at least three times.