Two microliters of the mix were recovered at the indicated time points and used for SDS-PAGE and western blotting
Two microliters of the mix were recovered at the indicated time points and used for SDS-PAGE and western blotting. When dephosphorylation of Arpp19 mutants was assessed in ATP-devoid extracts by autoradiography, 1?l of the in vitro 33P-labelled GwlK72M-phosphorylated Arpp19 mutants were supplemented with 9?l of Tris, 50?mM EDTA, 10?mM buffer and mixed with 10?l of ATP-devoid extracts an incubated at RT.Two-microlitre samples were then recovered at the indicated time points and submitted to western blot and autoradiography. show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division. oocytes. PKA-dependent phosphorylation of Arpp19 on residue S109 is essential to arrest oocytes in prophase I of meiosis18. Accordingly, the injection of a S109D phospho-mimetic Arpp19 form to these oocytes blocks meiotic resumption induced by progesterone (PG). How phospho-S109 Arpp19 negatively regulates meiotic DHBS resumption is completely unknown. In this work, we identify the major molecular determinants controlling the DHBS PP2A-B55 inhibitory activity of Arpp19. Moreover, we elucidate the mechanisms by which phospho-S109 controls S67 Arpp19 phosphorylation and, thus, PP2A-B55 inhibitory activity. Finally, we have discovered a double feedback loop between these two phospho-sites, which would be required to coordinate the proper temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation, hence ensuring a correct progression through meiosis and mitosis. Results DSG residues control Arpp19 dephosphorylation and PP2A-B55 binding Arpp19 is an intrinsically disordered protein that potently inhibits PP2A-B55. Despite its major role in the control of cell cycle progression3C6,19, little is known about the mechanisms by which this protein modulates phosphatase activity. The binding and the inhibition of PP2A-B55 by Arpp19 require its phosphorylation at S67 by Gwl. This site is located in a motif highly conserved from yeast to humans (QKYFDSGDY), which we will refer to from now as the DSG motif3,16,20,21. Once phosphorylated, Arpp19 acts as a highly competitive substrate that tightly binds PP2A-B55 but is slowly dephosphorylated by this phosphatase16. Why S67 dephosphorylation of Arpp19 is so slow is unknown. We sought to determine the properties contributing to the inhibitory activity of Arpp19. To this end, we used the well established cell-free extract system of oocytes. In this model, we measured the three major properties of this inhibitor as follows: (1) Gwl site dephosphorylation kinetics, (2) binding to PP2A-B55 and (3) physiologic capacity to DHBS promote mitotic entry. Previous data propose that the dephosphorylation of ENSA, another member of the endosulfine family, is modulated by the presence of basic KR residues flanking the DSG motif. These residues would be a PP2A-B55 recognition signal that would promote binding to PP2A-B55 and would ensure DHBS timely ENSA dephosphorylation22. To determine whether dephosphorylation and binding of Arpp19 to PP2A-B55 are modulated by basic KR residues, we mutated into alanine three of the basic aminoacids flanking the DSG motif of Arpp19 isoform 1 cloned from Stage VI oocytes. This isoform displays four additional aminoacids at positions 62C65 and, consequently, residue numbering will differ accordingly in the present study. We constructed a triple alanine mutant of the K36/K38/R40 region (named hereafter as the KKR motif) (Fig.?1A), which was further phosphorylated in vitro by recombinant Gwl and P33ATP. Phosphorylated wild type and triple KKR mutant Arpp19 were then supplemented to interphase egg extracts that were devoid of ATP to maintain kinases inactive (hereafter called kinase-inactivated extracts). The dephosphorylation pattern of the Gwl site S67 (S71 in Arpp19 isoform 1) was then followed by autoradiography to directly measure phosphatase activity and the levels of Arpp19 by western blotting (Fig?1B and Supplementary Fig.?1A). S67/S71 dephosphorylation of wild-type Arpp19 started at 8?min upon its addition to kinase-inactivated extracts, gradually decreased and fully disappeared at 30?min (Fig.?1B and Supplementary Fig.?1A). This dephosphorylation is catalysed by PP2A-B55, as it was fully blocked upon B55 depletion (Fig.?1B). Dephosphorylation of S67/S71 in the K36A/K38A/R40A (KKR/AAA) triple mutant was moderately delayed compared to wild-type Arpp19 and Rabbit Polyclonal to CRMP-2 (phospho-Ser522) a complete dephosphorylation was only observed at 40?min (Fig.?1B and Supplementary Fig.?1A). This data indicates that these basic residues would positively modulate the dephosphorylation of S67/S71 on Arpp19. Open in a separate window Fig. 1 Basic residues close to the DSG motif positively modulate S67/S71 dephosphorylation of Arpp19.A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity.