Journal: Nature Communications
Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop
doi: 10.1038/s41467-021-23657-0
Figure Lengend Snippet: A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.
Article Snippet: The antibodies used in this study are the following: Rabbit Polyclonal anti-Human Gwl , Rabbit Polyclonal Phospho-Cdc2 (Tyr15) (Cell Signaling Technology Cat#9111), Rabbit Polyclonal anti- Xenopus Arpp19 , Rabbit Polyclonal anti-PP2A/B55δ (Cell Signaling Technology Cat#2290), Rabbit Polyclonal anti- Xenopus Cdc27 , Rabbit Polyclonal anti- Xenopus Cyclin B2 , Rabbit Polyclonal anti- Xenopus Cdk1 , Rabbit Monoclonal PhosphoThr320 of PP1 (Abcam Cat#62334), Mouse Monoclonal Phospho-Erk (Cell Signaling Cat# 9106 S), Rabbit Polyclonal anti-phosphorylated Arpp19 (S67) (Cell Signaling Cat#5240 S), Rabbit Polyclonal anti-PRC1 (Santa Cruz Cat# 376982), Goat Polyclonal anti-phosphorylated PRC1 (T481) (Santa Cruz Cat#11768), Mouse Monoclonal anti-PP2A (C) subunit-α (isoform Merck Millipore Cat#05-42), Rat Polyclonal PP2A (A) subunit (Cell Signaling Cat#2260), Mouse Monoclonal anti-PP1 kindly gifted by Dr M Bollen and used in Ma et al. , Anti-PP4c (Bethyl Cat#A300-835A), Anti-PP6 (Santa Cruz Cat#393294), Goat anti-rat IgG-horseradish peroxidase (HRP) (Santa Cruz Cat#2006), HRP-conjugated anti-Rabbit secondary antibodies (Cell Signalling Technology Cat#7074), Donkey anti-goat IgG-HRP (Santa Cruz Cat# sc-2020), Rabbit Polyclonal anti-phosphorylated Arpp19 (S109/S113) (this study), and Rabbit Polyclonal anti-PP2A (B56) γ-subunit (this study).
Techniques: Mutagenesis, De-Phosphorylation Assay, In Vitro, Phospho-proteomics, Western Blot, Autoradiography, Staining
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![A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6004/pmc08196004/pmc08196004__41467_2021_23657_Fig6_HTML.jpg)