The phosphorylation of ARPP19 by Greatwall renders the autoamplification of MPF independent of PKA in Xenopus oocytes

Abstract

Entry into mitosis or meiosis relies on the coordinated action of kinases and phosphatases that ultimately leads to the activation of Cyclin B-Cdk1, also called MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest tightly controlled by a high PKA activity. Reentry into meiosis depends on Cdk1 activation that obeys a two steps mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein synthesis-dependent manner; then a regulatory network called MPF auto-amplification loop is initiated. This second step is independent of PKA and protein synthesis. However, none of the molecular components of the auto-amplification loop identified so far acts independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiological intact cell system, the Xenopus oocyte, we show that the phosphorylation of ARPP19 at S67 by the Greatwall kinase promotes its binding to the PP2A-B55 δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and plays an essential role within the Cdk1 auto-amplification loop for entry into the first meiotic division. Moreover, once phosphorylated by Greatwall, ARPP19 escapes the negative regulation exerted by PKA. It also promotes MPF activation independently of protein synthesis, provided a small amount of Mos is present. Taken together, these findings reveal that PP2A-B55δ, Greatwall and ARPP19 are not only required for entry into meiotic divisions, but are also pivotal effectors within the Cdk1 auto-regulatory loop responsible for its independence toward PKA negative control.

ARPP19 Phosphorylations by PKA and Greatwall: The Yin and the Yang of the Cell Decision to Divide

“…We also generated a nonphosphorylatable ARPP19 mutant at S67 by mutating S67 into an alanine (S67A-ARPP19). The S67A-ARPP19 mutant proved to be a strong inhibitor of meiosis resumption in response to either progesterone or PKI injection but also following the overexpression of constitutive active Gwl [110]. These results clearly demonstrate that ARPP19, once phosphorylated by Gwl at S67, is involved in the Cdk1 autoamplification loop by directly inhibiting PP2A-B55δ (Figure 7).…”

“…Cdc25 to launch the loop [70,103,105,109,110]. In contrast, PKA activity does not prevent the autoamplification loop when triggered by the inhibition of PPPs [17,104].…”

“…We showed that ARPP19 is phosphorylated at S67 by Gwl at the time of Cdk1 activation. Once phosphorylated by Gwl, ARPP19 interacts with PP2A-B55δ [110]. We then produced in vitro a Gwl-thiophosphorylated form of ARPP19 protein, which is resistant to dephosphorylation.…”

“…We then produced in vitro a Gwl-thiophosphorylated form of ARPP19 protein, which is resistant to dephosphorylation. Injecting this constitutively phosphorylated form of ARPP19 at S67 promoted meiosis resumption independently of progesterone and of the synthesis of either Mos or cyclin B [110]. We also generated a nonphosphorylatable ARPP19 mutant at S67 by mutating S67 into an alanine (S67A-ARPP19).…”

Meeting report – Oocyte maturation and fertilization: lessons from canonical and emerging models

“…Using frog oocytes, Olivier Haccard (CNRS, UPMC UMR7622, Paris, France) demonstrated that the phosphorylation of ARPP19 by Greatwall, which is controlled by basal levels of Cdk1, promotes the binding of ARPP19 to PP2A-B55, thereby inhibiting PP2A. This phosphorylation event renders the MPF auto-amplification independent of PKA (Dupré et al, 2013). The involvement of Greatwall and ARPP19 in MPF activation is conserved between starfish and frog, and it will be interesting to assess whether they also participates in MPF autoamplification in other species, such as invertebrates (Cnidarian or C. elegans) or simple marine chordates (urochordates).…”