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

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 found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) 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 MI. 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.


Introduction
Animal reproduction requires that oocytes undergo a specialized cell cycle called meiosis, in which two functionally specialized divisions occur in rapid succession to reduce genome ploidy. Following fertilization, the oocyte must then transition to a third and distinct division strategy, mitosis, for early embryonic development. This oocyte-toembryo transition occurs in a short temporal window, but must achieve high fidelity to ensure that heritable information is accurately transmitted from the parents to the developing embryo. At the center of this progression is a suite of cell cycle regulatory proteins and molecular machines that drive and integrate processes such as chromosome segregation, fertilization, and pronuclear fusion. An important goal is to unravel the complex regulatory mechanisms that precisely coordinate these divisions in time and space within the oocyte. It particular, it remains unknown how phosphorylation and dephosphorylation drive the meiotic divisions allowing oocytes to rewire the cell division machinery at the meiosis I/II transition to facilitate differing requirements.
The female meiotic cell cycle is distinct from mitosis in several ways, necessitating a unique regulatory control. First, oocytes remain in an extended primary arrest in a cell cycle state termed prophase I until receiving an extrinsic hormonal signal (Conti and Chang, 2016;Jaffe and Norris, 2010;Kishimoto, 2018;Von Stetina and Orr-Weaver, 2011). The meiotic divisions use a small, asymmetrically-positioned spindle to partition chromosomes into polar bodies, which do not contribute to the developing embryo (Severson et al., 2016). In addition, the first meiotic division segregates bivalent pairs of homologous chromosomes, whereas for meiosis II, this configuration is reversed and instead sister chromatids are segregated (Fig. 1A) (Watanabe, 2012). Finally, meiosis lacks a DNA replication phase between the polar body divisions, which enables the reduction of ploidy to haploid. How the cell division machinery is specialized to perform the distinct functions of meiosis I (MI), and then is rapidly reorganized for the unique requirements of meiosis II (MII) while remaining in meiosis and not exiting into gap or Sphase is an important open question.
In this study, we define phosphoregulatory mechanisms that drive the MI/MII transition. We undertook a proteomic and phosphoproteomic strategy using oocytes of the sea star Patiria miniata. Prior analyses have revealed proteome-wide changes in animal models including Xenopus, Drosophila, and sea urchins (Guo et al., 2015;Krauchunas et al., 2012;Presler et al., 2017;Zhang et al., 2019). However, the biology of these organisms limits access to a comprehensive series of time points spanning prophase I through the embryonic divisions including the critical meiosis I/II transition.
Instead, our sea star proteomics dataset spans the entire developmental window from Prophase I arrest, through both meiotic divisions, fertilization, and the first embryonic division (Fig. 1A) (Swartz et al., 2019). We identify a surprising differential behavior between serine and threonine dephosphorylation at the MI/MII transition that we propose underlies key regulatory differences between these meiotic divisions. This regulatory switch is driven by PP2A-B55, which is reactivated after MI to preferentially dephosphorylate threonine residues, thereby creating temporally distinct reversals of Cyclin-dependent kinase and Map kinase phosphorylation. We propose a model in which usage of threonine versus serine endows substrates with different responsivity to a common set of kinases and phosphatases, temporally coordinating individual proteins with meiotic cell cycle progression to achieve specific behaviors for MI and MII without exiting from meiosis.

Proteomic analysis reveals stable protein abundance during the oocyte-to-embryo transition
The oocyte-to-embryo transition involves dramatic changes in cellular organization, with an ordered series of events including fertilization, chromosome segregation, polarization, and cortex remodeling. To determine the basis for these cellular changes, we analyzed the proteome composition during the oocyte-to-embryo transition using quantitative tandem mass tag multiplexed mass spectrometry (Thompson et al., 2003). We obtained Prophase I-arrested oocytes from the sea star Patiria miniata and treated them with the maturation-inducing substance 1-methyladenine (1-MeAd) to trigger the resumption of meiotic progression in seawater culture (Kanatani et al., 1969). P. miniata oocytes progress through meiosis with high synchrony, with key events occurring at stereotypical times following 1-MeAd addition (Fig. 1A,S1A). Leveraging these features, we cultured isolated oocytes and collected biological triplicate samples at the following stages: 1) Prophase I arrest (Pro), 2) germinal vesicle breakdown (GVBD), 3) metaphase of meiosis I (MI), 4) prometaphase of meiosis II (MII), 5) just prior to pronuclear fusion (2-PN), and 6) metaphase of the first embryonic cleavage (FC) (Fig. 1A,S1A).
We first tested whether protein abundance changes could regulate the oocyte-toembryo transition (Fig. 1B). We identified 8026 total proteins, of which 6212 were identified in two independent time-course series, and 4635 in all three series (Supp.  1B). Surprisingly, we identified few proteins that changed in abundance during these different stages (Fig. 1C). In fact, 98.8% of proteins displayed a maximum fold-change of less than 2 from prophase I to the first embryonic cleavage, with 74.8% of proteins displaying less than a 1.2-fold change (Fig. 1D). The absence of changes in protein levels was not due to a bias in our analysis as protein abundance followed a normal distribution (Fig. 1E). Despite broad overall stable protein levels, there were several notable exceptions (Fig. 1F). For instance, Cyclin B levels were high in Prophase I, GVBD, and MI, but declined sharply in MII, before being partially restored in the first cleavage stage (Fig. 1F). These dynamics are consistent with APC/C-mediated destruction of Cyclin B during cell cycle progression (Evans et al., 1983;Okano-Uchida et al., 1998). Gene ontology analysis (Liao et al., 2019) of significantly regulated proteins revealed an enrichment in cytoskeletal proteins, protein involved in RNA binding, and ribosomal components (Supp . Table 2). Thus, although some critical regulators of cell cycle progression and other processes fluctuate in their levels, the sea star proteome is broadly stable during the oocyte-to-embryo transition.

New translation of selected proteins is required for meiotic progression
Although protein levels are largely constant across the oocyte-to-embryo transition, de novo translation could act to maintain steady state levels or may be required to produce a limited set of factors involved in meiotic progression. To test this premise, we globally prevented translation with the 40S ribosomal inhibitor emetine. Emetine-treated oocytes responded to 1-MeAd stimulation to initiate meiosis I, consistent with prior work (Houk and Epel, 1974), but instead of progressing to meiosis II, the maternal DNA decondensed and formed a pronucleus precociously, where they remained arrested even when control oocytes were initiating first cleavage ( Fig. 2A,B). Based on proteomics of emetine-treated meiosis II or pronuclear stage oocytes, we found that only 108 out of 7,610 proteins identified in our analysis significantly changed with emetine treatment (Supp . Table 3, Supp. Fig. 2A,B). These emetine-sensitive proteins fell into diverse categories, but were overrepresented for cytoskeletal elements and actomyosin-related proteins (Supp . Table   4). In summary, most proteins are insensitive to translational inhibition, indicating a general lack of turnover between MI and MII, but new protein synthesis is required for progression past meiosis I.
The requirement of protein synthesis for meiotic progression could reflect the need to translate select cell cycle factors. To test whether established cell cycle regulators must be translated de novo, we used morpholino injection to specifically prevent new translation of Cyclin B, one of the few proteins that varies in abundance (Fig. 2C,D), as well as Cyclin A, which is synthesized in late MI in a related sea star species (Okano-Uchida et al., 1998). We stimulated oocytes with 1-MeAd immediately following morpholino injection to ensure that pre-existing cyclin protein was unaffected. When new Cyclin A synthesis was blocked, oocytes underwent both meiotic divisions normally and the maternal and paternal pronuclei fused, but these zygotes then arrested with a single, fused pronucleus and failed to progress to the first cleavage (Fig. 2C,D). This is consistent with a role for Cyclin A in mitotic entry in cultured cells (Pagano et al., 1992). In contrast, oocytes injected with translation-blocking morpholinos targeting Cyclin B proceeded through MI normally, but failed to extrude the second polar body in MII and thus retained an additional centriole. Surprisingly, these oocytes successfully underwent pronuclear fusion, entered the first cleavage, and formed a mitotic spindle. This suggests that Cyclin B must be translated de novo following Anaphase I to drive meiosis II, but is dispensable for the initial transition from meiosis to embryonic mitosis. Finally, when we simultaneously prevented the new translation of both Cyclin A and B, oocytes completed meiosis I and then arrested in a pronuclear-like state without conducting meiosis II (Fig.   2C,D), similar to the effect of translational inhibition by emetine ( Fig. 2A,B). Taken together, our results suggest that the proteome during the oocyte-to-embryo transition is highly stable, but that the de novo translation of cyclins is required for meiotic progression.

Defining the phosphorylation landscape of the oocyte-to-embryo transition
The two meiotic divisions, fertilization, pronuclear fusion, and the first mitotic cleavage all occur within less than 3 hours in the absence of substantial changes in protein abundance ( Fig. 1C,D). This suggests that there are alternative mechanisms to rapidly re-organize the cell division apparatus during these transitions. Therefore, we assessed the role of phosphorylation across meiosis and the first mitotic cleavage (Fig. 3A). Our analysis identified a total of 25,228 phosphopeptides across three multiplexed time courses.
Hierarchical clustering of the dynamic phosphorylation behavior from prophase I to the first embryonic cleavage revealed several striking transitions in global phosphorylation status (Fig. 3B). First, Prophase I-arrested oocytes are distinct from the other stages in that they not only display a limited number of phosphorylation sites at the relative maximum phosphorylation levels (Fig. 3C), but also have the lowest overall phosphorylation state of the samples tested (Fig. 3D). Second, more than half of the total phosphorylation sites identified were maximally phosphorylated in MI, whereas phosphorylation was substantially reduced for MII (Fig. 3C). These patterns of phosphorylation imply a critical role for phosphoregulation in specializing the two meiotic and first cleavage divisions, and suggest a role for a low phosphorylation state in maintaining the prophase I arrest.

Maturation-promoting kinase activity across the oocyte-to-embryo transition
To remain arrested in Prophase I, Cyclin-dependent kinase 1 (Cdk1) and maturation promoting kinases must kept inactive. As our proteome analysis indicated that the majority of proteins in the oocytes, including kinases, are present constitutively ( Fig.   3B,E,G), kinase activity must be controlled post-translationally. Therefore, we next analyzed the pattern of phosphorylation events on established cell cycle kinases. The meiotic divisions and secondary arrest that occurs in the absence of fertilization require MAP kinase activity downstream of the conserved activator Mos (Dupré et al., 2011;Tachibana et al., 2000). Based on our phosphoproteomics and Western blotting, we found that a conserved activating phosphorylation on the MAP kinase p42/ERK phosphorylation (Y204) was undetectable in Prophase I-arrested oocytes, but high in meiosis I and II ( Fig.   3E,F). Although phosphorylation is low globally in Prophase I-arrested oocytes, we found high phosphorylation for inhibitory sites on Cdk1 or Cdk2 (Y21 or Y15, respectively) based on our phosphoproteomic analysis and Western blotting using phospho-specific antibodies against these conserved sites (Fig. 3G, H). The pattern of these phosphorylation events suggests that p42/ERK and Cdk are inactive in Prophase Iarrested oocytes, but are high throughout the meiotic divisions. Finally, meiotic resumption in sea star oocytes requires Greatwall kinase (Kishimoto, 2018). Greatwall is sequestered in the germinal vesicle in Prophase I sea star oocytes and is activated downstream of Cdk1/Cyclin B (Hara et al., 2012). We identified a conserved activating phosphorylation within the activation segment of Greatwall kinase (T194 in humans; T204 in sea star) (Supp. Fig. 3A) (Blake-Hodek et al., 2012;Gharbi-Ayachi et al., 2010), indicative of high Greatwall kinase activity during GVBD and MI, and reduced activity in later stages. In summary, our phosphoproteomic time-course reveals orchestrated transitions in the activity of regulatory kinases during the oocyte-to-embryo transition.

Prophase I arrest is enforced by high phosphatase activity
Our phosphoproteomics analysis indicated that Prophase I is characterized by low global phosphorylation. To determine whether this state is reinforced by phosphatase activity to maintain the primary arrest, we analyzed our dataset for modifications to the major cell cycle phosphatases, PP1 and PP2A (Nasa and Kettenbach, 2018), and their regulators. and of its regulatory subunit NIPP1 (Supp. Fig. 3C-E). PP1 T316 (corresponds to T320 in humans) is notably hypo-phosphorylated in Prophase I-arrested oocytes (Supp. Fig.   3B; (Beullens et al., 1999;O'Connell et al., 2012)), which would result in high PP1 activity.
Similarly, NIPP1 phosphorylation is low in Prophase I-arrested oocytes (Supp. Fig.   3C,D). Preventing NIPP1 phosphorylation with phospho-inhibitory mutations (P. miniata NIPP1 S199A or S197A S199A double mutants) resulted in a >5-fold increase in their binding to human PP1 when expressed in human 293T cells (Supp. Fig. 3F). Thus, the low NIPP1 phosphorylation in Prophase I oocytes would increase the association between PP1 and NIPP1 to modulate its phosphatase activity towards specific substrates.
We also identified phosphorylation events predicted to inhibit PP2A activity through its B55 subunit (Supp. Fig. 3G). Upon phosphorylation of ARPP19 by Greatwall kinase, phospho-ARPP19 (S67 in human, S106 in P. miniata) binds to and inactivates the regulatory PP2A regulatory subunit B55 (Gharbi-Ayachi et al., 2010;Okumura et al., 2014). Indeed, we found that PmARPP19 thio-phosphorylated in vitro by Greatwall kinase (Gharbi-Ayachi et al., 2010) resulted in increased Cdc25 phosphorylation when added to mitotic human cell lysates (Supp Fig. 3H). Thus, phospho-ARPP19 S106 acts an active B55 inhibitor such that ARPP19 phosphorylation can serve as a proxy for assessing PP2A-B55 activity in sea star oocytes. PmARPP19 S106 phosphorylation is low in Prophase I-arrested oocytes, but is high in GVBD and MI, before decreasing again in MII (Supp. Fig. 3I). Therefore, we conclude that PP2A-B55 activity is high in prophase I, low for MI, but reactivated following the MI/MII transition.
To test the functional requirement for these phosphatases in maintaining prophase I arrest, we treated oocytes with the potent PP1 and PP2A inhibitor Calyculin A. Following Calyculin A addition, we found that 100% of oocytes spontaneously underwent GVBD within 70 minutes (Supp. Fig. 4A; also see (Tosuji et al., 1991)). However, the oocytes failed to progress past this GVBD-like state based on the absence of a contractile actin network, chromosome congression, or spindle formation (Supp. Fig. 4B). Therefore, while phosphatase activity is required to maintain a normal prophase I arrest, its inhibition is not sufficient to recapitulate physiological meiotic resumption. Calyculin A treatment resulted in the broad upregulation of phosphorylation based on a phosphoproteomics analysis (Supp . Table 5), with 85% of phosphorylation sites being maximally phosphorylated after 70 min following Calyculin A treatment (Supp. Fig. 4C). Indeed, the phosphorylation events associated with the different meiotic stages increased, whereas those associated with the Prophase I arrest instead decreased (Supp. Fig. 4D).
Collectively, with phosphoproteomic and functional analyses indicates that there is high activity of PP1 and PP2A-B55 in Prophase I-arrested oocytes that enables the low global phosphorylation state, with additional waves of phosphatase activity controlling subsequent phases of meiotic phosphorylation.

PP2A-B55 drives selective dephosphorylation at the MI/MII transition
During meiosis, oocytes must undergo two consecutive chromosome segregation events without exiting into an interphase state. These rapid divisions occur within 30 minutes of each other in the sea star, but they each achieve distinct functions. Therefore, some MIassociated phosphorylations must be reversed to allow progression to MII, while others must be maintained to remain in meiosis. Of the stages tested, MI displayed the largest number of sites with maximal phosphorylation (Fig. 3A, 4A), including a large number of sites with a TP or SP consensus motif indicative of CDK or MAPK-dependent phosphorylation. Hierarchical clustering of sites maximally phosphorylated in MI revealed three distinct clusters. Strikingly, a subset of sites sharply decreased in phosphorylation after MI (Fig. 4B, Cluster 2), whereas other sites remain phosphorylated during MII and the first embryonic cleavage (Fig. 4B, Cluster 3). A third cluster displayed intermediate dephosphorylation kinetics (Fig. 4B, Cluster 1). These differential behaviors could reflect a mechanism by which the meiotic divisions are specified and underlie the transition from MI to MII.
To determine the mechanisms controlling these behaviors, we analyzed the specific phosphorylation events that are eliminated after MI (Fig. 4B, Cluster 2) compared to those that are retained (Fig. 4B, Cluster 3). Motif analysis of Cluster 2 sites revealed that they predominantly occur on threonine with proline in the +1 position (TP sites).
Furthermore, we identified an enrichment for a basic amino acids starting in the +2 position (e.g. lysine and arginine) and a depletion of acidic amino acids (e.g. aspartic and glutamic acid) (Fig. 4C). This phosphorylation site preference is consistent with the consensus motif dephosphorylated by PP2A-B55 that we and others recently reported (Cundell et al., 2016;Kruse et al., 2020;McCloy et al., 2015). In contrast, MI-specific phosphorylation sites are depleted for serine and for downstream acidic residues. We next directly compared the phosphorylation of SP versus TP sites in the three clusters and found that TP phosphorylation declines more substantially than SP after MI (Fig. 4D).
Notably, we also observed that the nature of the amino acids in the +2 position of TP site determined its dephosphorylation. TP sites with a small non-polar or basic amino acid were significantly more dephosphorylated than TP with acidic amino acids or proline ( Fig.   4E, Supp. Figure 5A). On average, the mean phosphorylation of TPxK motifs detected by phosphoproteomics peaked in MI, but then declined for MII (Fig. 4F). In further support of this analysis, Western blots using antibodies against phosphorylated pTPxK sites indicated a peak in MI, followed by strong reduction in threonine phosphorylation in MII ( Fig 4G). Taken together, these data reveal unexpected behavioral differences for SP vs.
TP phosphorylation sites at the MI to MII transition, with threonine residues followed by basic or nonpolar amino acids being preferentially dephosphorylated. These dephosphorylation behaviors are consistent with regulation by PP2A-B55, suggesting that this phosphatase must be reactivated at the MI/MII transition.
PP2A-B55 inhibition is required for meiotic resumption from Prophase I into MI in response to hormonal stimulation (Hara et al., 2012;Okumura et al., 2014), but a role for PP2A-B55 at the MI/MII transition has not been defined. To determine the activation state of PP2A-B55 at the MI/MII transition, we assessed its conserved regulatory pathway.

Selective dephosphorylation by PP2A-B55 is required for the MI/MII transition
Our data are consistent with a model in which PP2A-B55 reactivation at the MI / MII transition drives the selective dephosphorylation of substrates to rewire the cell division machinery and distinguish these divisions. We therefore sought to test the functional contribution of PP2A-B55 to the MI / MII transition. As small molecule inhibition of PP2A also inhibits other phosphoprotein phosphatases and resulted in a failure in meiotic progression (e.g. Supp. Fig. 4B), we instead altered its specificity through mutations designed to disrupt B55 interactions with the downstream basic patch in its substrates (Cundell et al., 2016;Xu et al., 2008) (Fig. 5C). We generated mutations in complementary acidic residues in B55, changing these to alanine or creating charge swap substitutions of these sites to lysine. Compared to wild-type B55, ectopic expression of either of these mutants had a potent dominant effect on meiotic progression. Oocytes expressing the alanine mutant (DE/A) successfully assembled the meiosis I spindle and completed cytokinesis of polar body I. However, the meiosis II spindles did not form properly, displaying a ball-like morphology. Furthermore, anaphase and cytokinesis did not progress normally, resulting a reduced rate of successful polar body II extrusion (Fig.   5D,E). Oocytes expressing the charge-swap mutations (DE/K) displayed a more severe phenotype: the first meiotic spindle formed normally, but remained arrested, with the bivalent chromosomes maintaining cohesion and the co-oriented centromeres remaining fused, even at time points where wild-type control oocytes completed meiosis II. However, although normal anaphase I and cytokinesis did not occur, the spindle poles eventually separated, ultimately resulting in two semi-distinct spindles. Staining for Centrin2 revealed that pole fragmentation was due to the separation of the pair of centrioles in the spindle pole (Fig. 5E). Therefore, alterations to PP2A-B55 substrate specificity allow meiosis I events to occur, but prevent changes to the cell division machinery that occur at the MI/MII transition. We therefore propose that PP2A-B55 serves as essential regulator of the MI/MII transition by selectively dephosphorylating substrates to achieve exit from meiosis I, but retaining sites that must remain phosphorylated for meiosis II to occur.

Threonine-specific dephosphorylation is essential for the spatiotemporal control of PP2A-B55 substrates
The selective TP dephosphorylation at the MI/MII transition suggests a potential mechanism to encode MI or MII-specific functions directly into substrates. Evolving phosphorylation sites as higher or lower affinity substrates for PP2A-B55 may provide temporal control for individual protein behaviors in meiosis. We identified several conserved phosphorylation sites on PRC1 (T470 in humans, T411 in sea star), TPX2 (T369 in humans, T508 in sea star), and INCENP (T59 in humans, T61 in sea star) (Supp.  Hummer and Mayer, 2009;Jiang et al., 1998). Consistent with the reactivation of PP2A-B55, these substrates display a decrease in phosphorylation following MI. As proof of principle for this model, we focused on the chromosome passenger complex (CPC) subunit INCENP, which contains both stable serine phosphorylations as well as MIspecific threonine phosphorylations (Fig. 6A). During anaphase of mitosis, the CPC transitions from the inner centromere to the central spindle, where it is required for cytokinesis (Carmena et al., 2012;Kaitna et al., 2000). The relocalization of human INCENP is opposed by phosphorylation on T59 (T61 in sea star), and requires dephosphorylation of this residue by PP2A-B55 (Supp. Fig. 5D) (Goto et al., 2006;Hummer and Mayer, 2009). However, the localization dynamics and impact of phosphorylation are not well defined in meiosis. We find that phosphorylation on T61 sharply decreased following MI, whereas other proline-directed sites on INCENP remained phosphorylated throughout the oocyte-to-embryo transition (Fig. 6A).
Sequence alignment of sea star T61 with human INCENP T59 revealed the presence of a conserved downstream basic patch, supporting its potentially conserved dephosphorylation by PP2A-B55 (Fig. 6B).
To test the role of this INCENP threonine residue in meiosis I, we used GFP fusion constructs with either wild-type PmINCENP, or with T61 replaced with serine (T61S), which is typically considered to be a conservative change that would preserve protein function. Wild-type INCENP localized to centromeres in metaphase of meiosis I, but then relocalized to the central spindle at anaphase of Meiosis I. In contrast, INCENP(T61S)-GFP failed to translocate to the central spindle in anaphase of MI and remained at high levels at centromeres (Fig. 6C,D). The retention of INCENP at centromeres in anaphase I suggests that this residue remains phosphorylated at the MI/MII transition when substituted with serine, a lower affinity substrate for PP2A-B55. Thus, the usage of serine versus threonine, modulated by adjacent charged amino acids, can directly encode substrates with differential responses to a common set of kinases and phosphatases, enabling rewiring events at key cell cycle transitions. Through this paradigm, the behaviors of individual proteins may be temporally coordinated with meiotic cell cycle progression to achieve specific behaviors.

Discussion
In this work, we define an extensive program of phosphorylation changes during the oocyte-to-embryo transition, spanning the complete developmental window from Prophase I arrest to the first embryonic cleavage. Using TMT-based proteomics, phosphoproteomics, and functional approaches, we find that overall protein levels are stable, but that selected cell cycle regulators, including Cyclins A and B, must undergo new protein synthesis for progression through meiosis. In addition, we identify a complex landscape of phosphorylation and dephosphorylation that underlies this developmental period. We find that the Prophase I arrest is characterized by low overall phosphorylation, and that maintaining this arrest requires phosphatase activity. Strikingly, although serine and threonine residues are often considered as conserved and interchangeable, we observe distinct behaviors for TP and SP phosphorylation at the MI/MII transition, with threonine sites being preferentially dephosphorylated. Such differential dephosphorylation suggests a novel paradigm for the regulatory control of oocytes, which must rapidly transition between the two meiotic divisions for the specialized meiotic cell cycle.
Our analysis reveals that upon decrease in Cdk activity at the MI/MII transition, selective dephosphorylation of TP versus SP sites by PP2A-B55 is essential for meiotic progression. Examination of the TP sites dephosphorylated after MI identified an enrichment for basic amino acids starting in the +2 position, matching the known consensus for PP2A-B55 substrates (Cundell et al., 2016;Kruse et al., 2020). Moreover, we find that PP2A-B55 is re-activated at the MI/MII transition, based on phosphorylation signatures of its inhibitory pathway, including Greatwall and ARPP19, as well as conserved PP2A-B55 substrates. Our results are consistent with prior work reporting a decrease in Cyclin B abundance and dephosphorylation of Greatwall kinase following MI and during first cleavage (Hara et al., 2012). Thus, the temporal profile of PP2A-B55 activity leaves it poised to play an essential role in the MI/MII transition for specializing the meiotic cell cycle and division machinery.
Our work supports the emerging picture that threonine and serine phosphorylation is not interchangeable (Cundell et al., 2016;Deana et al., 1982;Deana and Pinna, 1988;Hein et al., 2017;Pinna et al., 1976), but instead represent an important regulatory mechanism to temporally control cellular processes. In cultured mitotic cells, threonine dephosphorylation is important for timely mitotic exit. For example, inhibitory phosphorylations on the APC/C regulator Cdc20 are conserved as threonines, whereas serine substitution mutants display delayed dephosphorylation and delayed Cdc20 activation (Hein et al., 2017). Our work shows that this differential dephosphorylation can provide not just a kinetic delay, but also can confer distinct behaviors for the two different The rapidity between the two meiotic divisions (only 30 minutes in sea stars) places a selective pressure for specific substrates to be promptly dephosphorylated. In contrast, other sites may need to remain phosphorylated to prevent exit into interphase, creating evolutionary pressure for conservation as serine. The ability to segregate heritable material through meiosis is essential for organismal fitness. We propose that evolution has fine-tuned substrate dephosphorylation by selecting for amino acid sequences that favor or disfavor the interaction with PP2A-B55, thereby enabling precise temporal coordination of events in the oocyte-to-embryo transition.

Experimental Model and Subject Details
Sea stars (Patiria miniata) were wild-caught by South Coast Bio Marine (http://scbiomarine.com/) and kept in artificial seawater aquariums at 15 °C. Intact ovary and testis fragments were surgically extracted as previously described (Swartz et al., 2019).

Ovary and Oocyte Culture
Ovary fragments were maintained in in artificial seawater containing 100 units/mL pen/strep solution. Intact ovary fragments were cultured this way for up to 1 week until oocytes were needed, with media changes every 2-3 days. Isolated oocytes were cultured for a maximum of 24 hours in artificial seawater with pen/strep. To induce meiotic reentry, 1-methyladenine (Acros Organics) was added to the culture at a final concentration of 10 μM. For fertilization, extracted sperm was added to the culture at a 1:1,000,000 dilution. For emetine treatments (Fig. 2), oocytes were pre-treated with 10 μM emetine (Sigma-Aldrich) for 30 minutes prior to hormonal stimulation.

Mass Spectrometry Sample Preparation
Oocytes were collected by centrifugation at 200 x g and resuspending pellets and washed one time with wash buffer (50 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM MgCl2, 100 mM KCl, 10% glycerol), pelleted with all excess buffer removed, and snap frozen in liquid nitrogen. Samples were lysed and proteins were digested into peptides with trypsin.
Oocyte pellets were lysed in ice-cold lysis buffer (8 M urea, 25 mM Tris-HCl pH 8.6, 150 mM NaCl, phosphatase inhibitors (2.5 mM beta-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium molybdate) and protease inhibitors (1 mini-Complete EDTA-free tablet per 10 ml lysis buffer; Roche Life Sciences)) and sonicated three times for 15 sec each with intermittent cooling on ice. Lysates were centrifuged at 15,000 x g for 30 minutes at 4˚C. Supernatants were transferred to a new tube and the protein concentration was determined using a BCA assay (Pierce/ThermoFisher Scientific). For reduction, DTT was added to the lysates to a final concentration of 5 mM and incubated for 30 min at 55˚C. Afterwards, lysates were cooled to room temperate and alkylated with 15 mM iodoacetamide at room temperature for 45 min. The alkylation was then quenched by the addition of an additional 5 mM DTT. After 6-fold dilution with 25 mM Tris-HCl pH 8, the samples were digested overnight at 37˚C with 1:100 (w/w) trypsin (Promega). The next day, the digest was stopped by the addition of 0.25% TFA (final v/v), centrifuged at 3500 x g for 15 min at room temperature to pellet precipitated lipids, and peptides were desalted. Peptides were lyophilized and stored at -80˚C until further use.
After 1 hr at room temperature, an aliquot was withdrawn to check for labeling efficiency while the remaining reaction was stored at -80°C. Once labeling efficiency was confirmed to be at least 95%, each reaction was quenched by addition of ammonium bicarbonate to a final concentration of 50mM for 10 minutes, mixed, diluted with 0.1% TFA in water, and desalted. The desalted multiplex was dried by vacuum centrifugation and separated by offline Pentafluorophenyl (PFP)-based reversed phase HPLC fractionation as previously described (Grassetti et al., 2017).
For phosphoproteomic analysis, ~4 mg of peptides were enriched for phosphopeptides using a Fe-NTA phosphopeptide enrichment kit (Thermo Scientific) according to instructions provided by the manufacture and desalted. Phosphopeptides were labeled with TMT reagent and labeling efficiency was determined as described above. Once labeling efficiency was confirmed to be at least 95%, each reaction was quenched, mixed, diluted with 0.1% TFA in water, and desalted. The desalted multiplex was dried by vacuum centrifugation and separated by offline Pentafluorophenyl (PFP)based reversed phase HPLC fractionation as previously described (Grassetti et al., 2017).

Construct Generation
Patiria miniata INCENP was identified using the genomic resources at echinobase.org and previously published ovary transcriptomes (Kudtarkar and Cameron, 2017;Reich et al., 2015). Wild-type INCENP was amplified from first strand cDNA reverse transcribed from total ovary mRNA. T61A, S, and D mutants were generated by overlap extension PCR. These cDNAs were then cloned into pCS2+8 as c-terminal GFP fusions (Gokirmak et al., 2012). Wild-type and mutant versions of B55 were synthesized (Twist Biosciences) and cloned into pCS2+8 with standard restriction enzyme methods.

Oocyte Microinjection
For expression of constructs in oocytes, plasmids were linearized with NotI to yield linear template DNA. mRNA was transcribed in vitro using mMessage mMachine SP6 and the polyadenylation kit (Life Technologies), then precipitated using lithium chloride solution.
Prophase I arrested oocytes were injected horizontally in Kiehart chambers with approximately 10-20 picoliters of mRNA solution in nuclease free water. B55 constructs were injected at 500 ng/μl, and INCENP constructs were injected at 1000 ng/μl. After microinjection, oocytes were cultured 18-24 to allow time for the constructs to translate before 1-methyladenine stimulation. Custom synthesized cyclin morpholinos, or the Gene Tools standard control, were injected at 500 μM immediately before 1-methyladenine stimulation (Gene Tools).

Immunofluorescence, Imaging, and Immunoblots,
Oocytes were fixed at various stages in a microtubule stabilization buffer as described For gene ontology analysis for proteins significantly changes in abundance during oocyte to embryo transition or emetine treatment, human homologs of Patiria miniata proteins were identified using NCBI's BLAST+. A custom R script was then used to filter the BLAST results to only high-confidence matches (E-value < .01) and to remove redundant matches. The resulting list analyzed using WebGestaltR to find enriched GO terms (Liao et al., 2019). Motif analysis for selected and deselected amino acids surrounding a phosphorylation site was performed using the Icelogo web interface.
Statistical analysis to determine population difference of dephosphorylation preferences at the MI to MII transition was performed using an unpaired nonparametric Kolmogorov-

Smirnov (KS) T test.
Graphing and statistical analyses involving the scoring of meiotic phenotypes (e.g. with an adjacent ROI to measure the background intensity of equivalent size by RawIntDen. The background measurements were then subtracted from the centromere or midzone measurement, and the resulting value was normalized to the selection area to allow comparison between inner centromere and midzone intensity. The crystal structure of the PP2A-B55 holoenzyme (Fig. 5A) was displayed using UCSF ChimeraX (Goddard et al., 2018).

Data and materials availability:
Raw MS data for the experiments performed in this study are available at MassIVE and ProteomeXchange. Plasmids generated from this study are available on request and will be deposited to Addgene. Light blue shading represents standard deviation.