The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.


Supplementary Figure 1
Legend. GST-Arpp19 is truncated in C-terminus during expression and purification from bacteria. (a) GST-Arpp19 coupled to sepharose GSH-beads was phosphorylated or not in metaphase II (MII) extracts. Proteins bound to beads were subjected to a 10% SDS gel electrophoresis. After Coomassie staining, two major bands were detected, the upper and most abundant one corresponding to the molecular weight expected for full-length GST-Arpp19 (FL-gst Arpp19) and a lower one that could correspond to a truncated form of Arpp19 (T-gst Arpp19). The preparative stained gel was prepared one time prior its analysis. kDa: kiloDalton. (b) Each band was cut out from the polyacrylamide gel and analyzed by LC-MS/MS.
The sequences of peptides identified by LC-MS/MS analysis from either FL-gst Arpp19 or T-gst Arpp19 are aligned with Xenopus Arpp19 sequence. The C-terminal peptide, PSLVASKLAG, is never recovered from T-gst Arpp19. When GST-Arpp19 has been phosphorylated in MII extracts, phosphorylated S109 (in red and underlined) is detected by LC-MS/MS in some peptides generated by FL-gst Arpp19 but never in the peptides generated by T-gst Arpp19. In contrast, phosphorylated S67 (in blue and underlined) is detected in peptides generated by both FL-gst Arpp19 and T-gst Arpp19. Source data are provided as a Source Data file. Method. Bacterially expressed GST-Arpp19 was bound to Sepharose GSH-beads 1 . GST-Arpp19 coupled to beads was incubated or not in metaphase II extracts to generate the double S167-S109 phosphorylated form of Arpp19. (https://www.ebi.ac.uk/pride/profile/reviewer_pxd022739).

Supplementary Figure 2
Legend. Biochemical isolation of S109-phosphatase from prophase extracts -Separation of fraction 6 from the Mono Q column with Phenyl-Superose and Superose 12 columns. (a) Prophase oocytes were lysed and centrifuged at 15,000 xg. The supernatant was then ultracentrifuged or not at 100,000 xg. S109-phosphatase activity was assayed in the 15,000 xg and 100,000 xg supernatants supplemented or not with PKI using pS109-GST-Arpp19 as a substrate (pS for phosphorylated substrate). S109 phosphorylation of GST-Arpp19 (pS109) and total GST-Arpp19 ( gst Arpp19) were western blotted using respectively phospho-S109-Arpp19 and GST antibodies. GST-Arpp19 coupled to beads was doubly phosphorylated and then used as a substrate of S109-and S67-phosphatase activities in prophase extracts as described in the Method section.
In experiment 3, S109-phosphatase activity was recovered in a single fraction after the Mono Q column, Q8, which was further loaded on the Phenyl-Superose column. S109-phosphatase activity was recovered in PS11 after the Phenyl-Superose column. PS11 was loaded on the Superose 12 column. The phosphatase content was estimated by LC-MS/MS sequencing in fractions from Superose 12 column. S109-phosphatase activity is indicated with "+" or "-". Data availability: The data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022739 experiment 3 -Analysis of PS11 and Superose fractions (input: Q8 > PS11). S109phosphatase activity was recovered in a single fraction after the Mono Q column, Q8, which was further loaded on the Phenyl-Superose column. S109-phosphatase activity was recovered in PS11 after the Phenyl-Superose column. PS11 was loaded on the Superose 12 column.
The phosphatase content was estimated by LC-MS/MS sequencing in PS11 and in fractions from Superose 12 column. S109-phosphatase activity is indicated with "+" or "-". Data availability: The data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022739