Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes

Abstract

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we find that protein levels are broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.

M-Phase oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

“…There are two probable causes of the abnormalities observed in oocytes treated with TMC for the duration of meiosis (0-16 h): (1) continued phosphorylation of prometaphase PP1 substrates, and (2) altered protein stability. Prometaphase PP1 substrates have been revealed in a recent study of meiotic phosphorylation dynamics in starfish oocytes, with a subset of CalA (PP1/PP2A inhibitor) sensitive protein phosphorylations peaking at NEBD and decreasing by metaphase I (Swartz et al, 2021). In oocytes with PP1 inhibition for the duration of meiosis (0-16 h), these early M-Phase PP1 phospho-sites could be maintained, likely contributing to the defects observed in these oocytes.…”

“…PP1 activation at G2/M transition (0-5 h post-meiotic arrest release) reduces oocyte viability significantly more than PP1 activation during M-Phase (3-8 h post-meiotic arrest release). Prophase I arrest (equivalent to G2 in mitosis) is characterized by high phosphatase activity and low kinase activity, particularly kinases that drive M-Phase (e.g., CDK1) (Swartz et al, 2021). Conversely, high kinase activity and low phosphatase activity maintains M-Phase progression.…”

“…Oscillations in phosphorylation of RIPPOs and PP1c, particularly phosphorylation of PP1c's inhibitory site (pT320 on PP1cα and the equivalent sites in PP1cβ and PP1cγ), during M-Phase play a critical role in the formation of PP1 holoenzymes and PP1 activity, and ultimately mitotic M-Phase progression (Dohadwala et al, 1994;Kwon et al, 1997;Nasa et al, 2018). Notably, oscillations in RIPPO and PP1c phosphorylations in M-Phase of starfish and mammalian oocytes mirror those observed in mitotic cells (Adhikari et al, 2012;Dohadwala et al, 1994;Kwon et al, 1997;Swartz et al, 2021).…”

“…This cyclic PP1 activity is driven in part by a CDK1-mediated inhibitory phosphorylation of PP1c (pPP1cα(T320) or equivalent sites in PP1cβ and PP1cγ) (Dohadwala et al, 1994;Kwon et al, 1997). The kinetics of PP1 inhibitory phosphorylation follows an identical pattern in starfish and mouse oocytes, suggesting similar fluctuations in PP1 activity during oocyte meiosis (Adhikari et al, 2012;Swartz et al, 2021). Therefore, we sought to determine if a reduction in PP1 activity is critical for meiotic resumption by culturing oocytes in PDP-Nal during the G2/M transition (0-5 h post-meiotic arrest release; Fig.…”

“…In mitosis, PP1 and PP2A have unique temporal and spatial regulation, and work in concert to oppose CDK1 activity, preventing M-Phase entry and promoting metaphase-to-anaphase transition (Bancroft et al, 2020;Grallert et al, 2015;Ma et al, 2016;Rogers et al, 2016). In mammalian oocytes, inhibition of PP1/PP2A with reagents such as okadaic acid and Calyculin A causes major meiotic abnormalities, and oocyte-specific conditional loss of PP2A causes substantial defects in chromosome segregation and M-Phase progression (Alexandre et al, 1991;Gavin et al, 1991;Hu et al, 2014;Mailhes et al, 2003;Schwartz and Schultz, 1991;Smith et al, 1998;Swain et al, 2007;Swain et al, 2003;Tang et al, 2016;Wang et al, 2004). However, data also suggest that PP2A is not the only phosphatase that regulates meiosis in mammalian oocytes, and so we sought to elucidate PP1's functions in this important cell type, building on evidence that PP1 has important roles in meiosis in Drosophila, Caenorhabditis elegans, starfish oocytes, and mammalian spermatogenesis (Hattersley et al, 2016;Oppedisano et al, 2002;Swartz et al, 2021;Wang et al, 2019a).…”