Role of Protein Phosphatase-2A (PP-2A) in the Control of Mitosis and Meiosis.

A specific form of Protein Phosphatase-2A (PP-2A), namely PP2A-B55δ was proposed to occupy a central role in the control of mitosis entry and exit, and meiosis in Xenopus oocytes [1,3]. It was held that PP2A-B55δ is responsible for dephosphorylating substrates of cdc2/Cdk1 and that inhibition of PP2A-B55δ by Arpp-19 phosphorylated at serine 67 by Greatwall kinase triggers entry of both mitosis and meiosis in Xenopus oocytes. It was further declared that the phosphorylation of Arpp19 at serine 109 by PKA underlies the blockade of meiotic division and that dephosphorylation of serine 109 of Arpp19 triggers resumption of meiotic division in Xenopus oocytes [4]. Recently two groups have stated that PP2A-B55δ is the protein phosphatase that is responsible for dephosphorylating both serine 67 and serine 109 of Arpp19 [4,5] However, unfortunately for the authors concerned [1-5], no verifiable scientific evidence exists that shows that Arpp19 is a specific inhibitor of PP-2AB55ɗ when Arpp19 is phosphorylated at serine 67 by Greatwall kinase and that Arpp-19 phosphorylated at serine 67 and Arpp19 phosphorylated at 109 are both specifically dephosphorylated by PP-2AB55ɗ Arpp19. The idea that Arpp-19 phosphorylated at serine 67 is both an inhibitor and a substrate of PP-2AB55ɗ has more to do with science fiction than science. The role of other Protein Phosphatases, including, PP-2A-B'56ɗ and Protein Phosphatase-1
 I (PP-1
 I ) cannot be ignored. Evidenceis presented and discussed here..

B55ɗ and the B'56ɗ of PP-2A-B'56ɗ are distinct gene products that are unrelated to one another with different properties and phosphorylation sites). In their paper entitled "Greatwall phosphorylates an Inhibitor of protein phosphatase-2A that is essential for mitosis" published in Science [Science (2010) Vol 330, pp1670-1673], the authors, Moshida, S., Maslen, S.L., Skehel, M. and Hunt, T., purported to show that α-Endosulfine (Ensa), a molecule that was homolohous and had identity with Arpp19, was an inhibitor of protein phosphatase-2A1 that contains the B55ɗ subunit (PP-2A-B55ɗ) when Ensa is phosphorylated by Greatwall kinase at serine 67 and that the specific inhibition of PP-2A-B55ɗ by Ensa underlies in large part entry into mitosis in Xenopus oocytes. While the proposal appears quite attractive, unfortunately evidence showing that PP-2A-B55ɗ is the major protein phosphatase that dephosphorylates substrates of Cdk1, including Wee1, Myt1 and Cdc25 was lacking. Evidence that Ensa Phosphorylated by Greatwall kinase at serine 67 was a specific inhibitor of PP-2A-B55ɗ was also not very convincing.
In the paper entitled "Greatwall phosphorylates an Inhibitor of protein phosphatase-2A that is essential for mitosis" and published in Science [Science (2010) Vol 330, pp1670-1673] , the authors, Moshida, S., Maslen, S.L., Skehel, M. and Hunt, T. started with the assumption that PP-2A-B55ɗ was the major protein protein phosphatase that was responsible for the dephosphorylation of Cdk1 substrates and that depletion of PP-2A-B55ɗ accelerates mitotic progression in Xenopus extracts by referencing a previous paper of theirs [3] to bolster their assumption which was not correct (See an Investigative Critique of the paper in [109]. and Hunt, T. claimed that: (i) after depleting the protein phosphatase activity in extracts of Xenopus oocytes in interphase with anti-PP1C antibody, almost 70% of the protein phosphatase activity remained, (ii) after depleting the protein phosphatase activity with anti-PP2A-A antibody, almost no protein phosphatase activity remained, and (iii) after depleting the protein phosphatase activity with anti-PP5 antibody almost 85% of the protein phosphatase activity remained. The authors, Mochida, S., Ikeo, S., Gannon, J. and Hunt, T. did not seem to be able to do simple arithmetic because the above results imply that at least ~45% of the total protein phosphatase activity using their model substrate was due to PP-1 and PP-5 and that the rest was due to PP-2A. Simple arithmetic dictatesd that after depletion with anti-PP2A-A antibody, at least ~45% of the total protein phosphatase activity should still be measurable but somehow mysteriously disappeared.
What happened to the ~45% of the total protein phosphatase activity? In Figure 2, The authors, Mochida, S., Ikeo, S., Gannon, J. and Hunt, T. showed that after depletion with anti-PP2A-A antibody, only 5% of the total protein phosphatase activity remained. Only after depletion with anti-PP-1C, anti-PP5 and anti PP-2A-A antibodies together, would one see almost no protein phosphatase activity in extracts of Xenopus oocytes at interphase. Perhaps more importantly, the authors of the paper did not seem to know that a large proportion of PP-1 is in an inactive form termed PP-1I and requires phosphorylation on threonine 72 of its Inhibitor-2 moiety by PP-1I activating kinase (PP-1I ACK) to exhibit its full activity [110,111]. Had the authors determined the activity of PP-1I, they would have seen that more than 50% of the protein phosphatase activity in the extracts of Xenopus oocytes would be accounted for by spontaneously active PP-1 and PP-1I.
purporting to show that PP-2A-B55ɗ is the major protein phosphatase responsible for dephosphorylation of the model substrate in extracts of Xenopus oocytes at interphase is quite misleading and possibly dishonest. In Figure 3, the authors Mochida, S., Ikeo, S., Gannon, J. and Hunt, T. claimed that after depleting 65% of the PP-2A with the anti-Bɗ subunit antibody, they could measure ~35% of the total protein phosphatase activity in extracts of Xenopus oocytes at interphase. They should be measuring at least twice that amount. Again, the authors, Mochida, S., Ikeo, S., Gannon, J. and Hunt, T. did not appear to be able to do simple arithmetic. In view of the fact that according to the results of Figure 2 which showed that almost ~55% of the total protein phosphatase activity was accounted for by PP-1 and PP-5, only ~45% of the remaining total protein phosphatase could be accounted for by PP-2A-B55ɗ. According to a simple calculation (~45% of ~45%), the % of total protein phosphatase that could be depleted by anti-PP-2A-Bɗ should be no more than ~21%. What happened to the almost ~79% of the total protein phosphatase activity that should be measurable after depletion with anti-PP-2A-Bɗ?
Although, the authors, Mochida, S., Ikeo, S., Gannon, J. and Hunt, T. did not say how many times they performed the experiments described in Figure 3 (there was no statistics presented!), it can be said that the pronouncement that PP-2A-B55ɗ was the major protein phosphatase that dephosphorylated Cdk1 substrates is misleading at best and dishonest at worse.
In Figure  and Hunt, T. claimed that PP-2A-B55ɗ "controls the proper timing of mitosis and is essential for protein dephosphorylation in exit of mitosis". In Figure  It must be noted that the cloning of A and C subunits of PP-2A of Xenopus oocytes was performed by Bosch et al. [112] and these cDNA clones were used by the authors of the paper reviewed here. In the paper by Bosch et al. [112], the apparent molecular mass of the A subunit of PP-2A is described as 65 kDa on SDS PAGE whereas in the paper by Mochida et al. [3], the apparent molecular mass A subunit of PP-2A is described as less than 52 kDa. In the paper by Bosch et al. [112], the A subunit of PP-2A gave a doublet by Western Immunoblotting of extracts of Xenopus oocytes. The authors of the paper entitled "Regulated activity of PP-2A-B55ɗ is crucial for controlling entry into and exit from mitosis in Xenopus egg", published in EMBO Journal [EMBO. J. (2009) Vol. 28, pp2777-2785], did not give any information about the specific activity of reconstituted PP-2A-B55ɗ nor did they characterize the molecular mass of the trimeric enzyme. It is therefore not possible to determine whether the reconstituted PP-2A-B55ɗ described in the paper was indeed a trimeric holoenzyme complex and not just a mixture of the three subunits.
In Figure  and Hunt, T. provided no convincing evidence that PP-2A-B55ɗ was the major protein phosphatase that was responsible for dephosphorylating cdc2/Cdk1 substrates and was the only protein phosphatase that was essential for entry and exit of mitosis in Xenopus oocytes. It can be surmised that the experimental procedures, results and conclusions associated with the paper are at best misleading and at worse dishonest.
Let us get back to the paper of the authors Moshida, S., Maslen, S.L., Skehel, M. and Hunt, T. entitiled "Greatwall phosphorylates an Inhibitor of protein phosphatase-2A that is essential for mitosis", authored By Moshida, S., Maslen, S.L., Skehel, M. and Hunt, T., and published in Science [Science (2010) Vol 330, pp1670-1673] in which they purported to show that α-Endolfine (Ensa) was an inhibitor of protein phosphatase-2A1 that contains the B55ɗ subunit (PP-2A-B55ɗ) when Ensa is phosphorylated by Greatwall kinase at serine 67 and that the specific inhibition of PP-2A-B55ɗ by Ensa underlied in large part entry into mitosis in Xenopus oocytes. As stated above, the authors of the paper [1]started with the assumption that PP-2A-B55ɗ was the major protein phosphatase that was responsible for the dephosphorylation of cdc2/Cdk1 substrates and that depletion of PP-2A-B55ɗ accelerates mitotic progression in Xenopus extracts by referencing to their previous paper [3] which was quite misleading and could be dishonest (See above).
In Figure 2 of the paper [1], the authors, used reconstituted PP-2A-B55ɗ that was presumably prepared as described in [3] and showed that using a model substrate, 32  In Figure 2A of the paper [1], the authors showed that dephospho α-Endolfine (Ensa) had no effect on the activity of PP-2A-B55ɗ which is contradictory to the results of William et al. [7] which showed that dephospho α-Endolfine (Ensa) inhibited PP-2A-B55ɗ with an IC50 of ~500 nM. The authors of the paper [1] stated without reservation that thiophospho ser 67-α-Endolfine (Ensa) is a specific inhibitor of PP-2A-B55ɗ in contradiction to Andrade, E.C. et al. [113] who stated that ARPP16 interacted with the A subunit of PP-2A1 and that ARPP16 phosphorylated by microtubule-associated serine/threonine kinase 3 (MAST3 kinase) on serine 46 becomes an inhibitor of PP-2A containing the B55α and B'56ɗ subunits (Although, it must be noted that only 70% inhibition was achieved with 200 nM of thio-phospho-ARPP16. It is not clear why a full dose response inhibitory curve of % Control v. Concentration of thio-phospho-ARPP16 and phospho-ARPP16 to determine the IC50 of the Inhibition of PP-2As could not be performed. One of the authors of the paper, Angus C. Nairn ought to know better). Figure 2A of the paper [1] that showed that thiophospho ser 67--α-Endolfine (Ensa) inhibited PP-2A-B55ɗ by almost 90% is in contrast to the results of Gharbi-Ayachi, A. et al. [2] which showed that PP-2A complex obtained by immunoprecipitation from interphase extracts was inhibited by thio-phospho-GST-α-Endolfine (Ensa) by less than ~45% after 120 mins incubation (it is not clear why Gharbi-Ayachi, A. et al. [2] had to incubate the PP-2A complex with thio-phospho-GST-α-Endolfine (Ensa) for 120 mins.
If thio-phospho-GST-α-Endolfine (Ensa) is a specific inhibitor of PP-2A complex and the immunoprecipitated protein phosphatase is active, incubation of 10 mins or less should be sufficient) .
The authors of the paper [1] stated that thiophospho ser 67-α-Endolfine (Ensa) is a specific inhibitor of PP-2A-B55ɗ but did not study the effect of thiophospho ser 67- There are too many problematic data and statements in the paper by Williams, C.B. et al [7] to enumerate here.). As the concentration of α-Endolfine (Ensa) has been estimated be present at ~150 to 300 nM in Xenopus oocyte extracts compared to that of ~50 to 70 nM The paper by Mochida, S. [113] also showed that dephospho-α-Endolfine can inhibit PP-2A-B55ɗ with an IC50 of 1800 nM (If the data is to be believed as it appears that the author does not seem to know how to perform a protein phosphatase properly. In the paper by Mochida, S. [113], the author stated that in each protein phosphatase assay, he used 0.8 unit of enzyme (i.e that amount of enzyme that caused the release of 0.8 nmol phosphate per min and that the assay lasted 120 min. Simple arithmetic indicated that in his control assay, he was measuring the release of 96 nmol 32 P-Phosphate. The author of the paper must ask the question whether it is possible to measure the release of 96 nmol 32 P-Phosphate and still remain in the linear part of the protein phosphatase assay. To be able to measure 96 nM 32 P-Phosphate is more in the realm of science fiction than science. As the author did not state the specific radioactivity of the 32 P-labeled substrate, it is impossible to determine what he was measuring and how he was able to measure 96 nmol of 32 P-Phosphate).
The paper by Kim, M.-Y. et al [4] showed and stated in Figure 5C  al. [3] which is misleading at best and dishonest at worse [3,109]. Can one assay for PP-  Figure 5C. What is quite remarkable is that there is almost no inhibition of PP-2A from C. elegans by neither D WT and D 68A. In Figure 5D of the paper by Kim, M.-Y. et al [4], it was shown and stated that a protein phosphatase activity in Xenopus extract was apparently inhibited by Dm Endos S68D with IC50 ~1,5 µM.
First, the result did not show that the protein phosphatase being inhibited was a form of PP-2A. Second, with an IC50 of ~ 1.5 µM, there is no difference between inhibition by so called phospho-Endos mimetic (Dm Endos S68D) and dephospho-Endos that apparently inhibited PP-2A-Bα and PP-2A-Bɗ with IC50 of 0.5 µM and 0.8 µM respectively [7,113] casting doubt on the phosphorylation dependent inhibition of PP-2A by Endos. The work described in the paper [1] is in contradiction with the work of Rangone et al. [114] in which the authors stated that they were unable to demonstrate a substantial inhibitory effect of phospho-Endos upon the ability of PP-2A to dephosphorylate histone H1 phosphorylated by Cdk1. One of the authors's of the paper entitled "Greatwall phosphorylates an Inhibitor of protein phosphatase-2A that is essential for mitosis", and published in Science [Science (2010) Vol 330, pp1670-1673] was quoted as stating that " the use of other substrates to assess for the inhibition of PP-2A by phospho-Endos was apparently less effective". These results indicated that any effect that phospho-Endos might be having on a form of PP-2A was most probably substrate directed and not enzyme directed, and that phospho-Endos could not be a specific inhibitor of PP-2A-B55α or PP-2A-B55ɗ. It is significant that Rangone et al. [114] stated that "From a genetic perspective, it is noteworthy that we observed only weak zygotic interactions between Endos and twins mutant. For example, adding either tws allele to either Endos allele (as endos+/endos tws) rescues the eclosion delay and the mild cuticular phenotype but not the female sterility . . . ".
The results described in the above paper and other papers that subscribed to a key role of PP-2A-B55ɗ in the control of dephosphorylation reactions in mitotic entry and exit do not take into account the results of (A) Margolis et al. [115] which showed that (i) PP-1 was required to dephosphorylate serine 287 of Cdc25, (ii) the effect of PP-1 on cdc25 was antagonized by a specific inhibitor of PP-1, I-1, (iii) PP-1 forms a complex with cdc25, and (iv) dephosphorylation of serine 287 of cdc2 is masked by 14-3-3 during interphase, (B) Margolis et al. [116] which showed that (i) the dissociation of 14-3-3 and dephoshorylation of of serine 287 of Cdc25 by PP-1 was regulated by phosphorylation of The paper of Manchado et al. [118] which showed that suppression of B55α and B55ɗ prevented 60% and 40 % respectively of mitotic exit in Cdc20 null cells treated with Mast1 (mammalian Greatwall kinase) RNAi, suggesting that "both PP-2A-B55α and PP-2AB-55ɗ participated in the PP-2A functional complexes required for mitotic exit in Cdc20 null fibroblasts" and that there was no selectivity for PP-2AB-55ɗ as far as Greatwall kinase was concerned. (ii) The work of Schmitz et al. [119] which showed that while many forms of PP-2A were required at all stages of mitosis, PP-2A-B55α was involved in the exit of mitosis and rate limiting only for post-anaphase progression. (iii) The work of Hegarat et al. [120] which showed that while B55 and B55 depletions did cause an increase in metaphase Cdk substrates phosphorylation, they did not affect the relative amount of Cdk1 substrates dephosphorylation following Cdk1 inhibition, that  [36,120,121] or phosphorylation of the B, B' or B'' subunits [27,66,[121][122][123] to exhibit full activity. The most compelling evidence against inhibition of the PP-2A subfamily of enzymes as a normal form of cellular control during mitosis is the fact that multiple studies have shown that marked inhibition of PP2A would automatically lead to cell transformation and tumorigenesis [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100].

The paper of Gharbi-Ayashi et al [2].
In the paper entitled "The substrate of Greatwall kinase, Arpp19 controls mitosis by The only circumstantial evidence that Arpp19 and α-Endolsulfine could be inhibitors of PP-2A was presented in Figure 3 of the paper. In Figure 3A, it was shown that the A and C subunits of PP-2A was bound to Arpp19 and α-Endolsulfine in Interphase extracts and not Mitotic extracts of Xenopus oocytes. The rationale for analyzing only the A and C subunits of PP-2A in the pull-down of protein complexes with GST-Arpp19-Sepharose and GST-α-Endolsulfine-Sepharose was quite puzzling. The authors were presumably referring to the studies of Mochida et al. [3] in which the authors stated that "Gwl mediates entry into mitosis through the inhibition of PP2A". However, the reliance of the studies of Mochida et al. [3] was misplaced as the work of Mochida et al. can be construed as misleading at best and dishonest at worse [1,3,109]. Specifically, the authors of the paper by Mochida et al. [3] were unable to do simple arithmetic with respect to the contribution of PP-2A in extracts of Xenopus oocytes at interphase and did not prove that PP-2A-B55ɗ is the major protein phosphatase that dephosphorylates the keys substrates of Cdk1. The paper by Mochida et al. [1] also started by assuming that PP-2A-B55ɗ was the major protein phosphatase that was responsible for dephosphorylating "model CDK substrates" and that depletion of PP-2A-B55ɗ accelerates mitotic progression in Xenopus extracts". As reviewed in [109], the paper by Mochida et al. [3] suffers from many anomalies and inconsitencies.
In order to prove that α-Endolfine and Arpp19 phosphorylated at serine 67 by Greatwall incubation with the substrate, the authors showed ~25% of the total phosphate was released in the control experiment and that ~10% of the total phosphate was released in the experiments in which 5.3 µg of thio-phospho-GST-α-Endolfine or thio-phospho-GST-Arpp19 were added and incubated for 10 mins. If one was to correctly plot % of control v. concentration of added thio-phospho-GST-α-Endolfine or thio-phospho-GST-Arpp19, one would obtain a plot which showed that at 60 mins, there was ~40 % inhibition of the enzyme. After 120 mins incubation with the substrate, the authors showed ~32% of the total phosphate was released in the control experiment and that ~15% of the total phosphate was released in the experiments in which 5.3 µg of thiophospho-GST-α-Endolfine or thio-phospho-GST-Arpp19 was added and incubated for 10 mins with the enzyme. If one was to correctly plot % of control v. concentration of added thio-phospho-GST-α-Endolfine or thio-phospho-GST-Arpp19, one would obtain a plot which showed that at 120 mins, there was 46% inhibition of the enzyme. The result suggests that the amount of thio-phospho-GST-α-Endolfine or thio-phospho-GST-Arpp19 that were added (5.3 µg) was not enough to saturate all of the PP-2A in the assay as the maximal inhibition that is achieved was ~46% inhibition which is impossible and against the laws of nature . The result also suggests that the effect of thio-phospho-GSTα-Endolfine or thio-phospho-GST-Arpp19 could be substrate directed and not enzyme specific.
Can an experiment to prove specific phosphorylation dependent inhibition of PP-2A by thio-phospho-GST-α-Endolfine and thio-phospho-GST-Arpp19 be taken seriously when the maximal inhibition that could be achieved was less than 50% and one must practice mental acrobatics to actually see any inhibition. (v) In the legend to Figure 3D, the authors stated that Gels were scanned by a Typhoon Scanner and quantified with Image Software. What was exactly 100%. (vi) In the legend to Figure 3D  Following the publication of the papers by Mochida et al. [1] and Gharbi-Ayashi et al. [2], several copycat papers appeared to show that PP-2A-B55ɗ was also the protein phosphatase that was responsible for the control of meiosis in Xenopus oocytes [5,9,143] These paper purported to show that the stimulation of Xenopus oocytes arrested at prophase by progesterone resulted in or was accompanied by the inhibition of PP-2A-B55ɗ by Arpp19 phosphorylated by Greatwall kinase [5,9,143 ]. Since these copycat papers were based on the papers of Mochida et al. [1] and Gharbi-Ayashi et al. [2], they did not even bother to do the requisite experiments to show and prove that Arpp19

The paper by Dupre et al [5].
Extrapolating from the misleading and possible dishonest results of Mochida et al. [1] and Gharbi-Ayashi et al [2], the paper entitled "Phosphorylation of ARPP19 by Greatwall renders the amplification of MPF independent of PKA in Xenopus oocytes", and publshed in Journal of Cell Science [5], the authors, by Dupre, A., Buffin, E., Roustan, C., Nairn, A.C., Jessus, C., and Haccard, O. purported to show that phosphorylation of the ARPP19 by Greatwall Protein Kinase promoted its binding to and inhibited Protein phosphatase-2A (PP-2A) that contains the B55ɗ subunit and that this process that was controlled by Cdk1, had an essential role within the cdk1 auto-amplification loop for the meiotic division in Xenopus oocytes. The authors of the above paper suggested that "PP-2A-B55ɗ, Greatwall and ARPP19 are not only required for entry into meiotic division but are also pivotal effectors with the cdk1 auto-amplification loop responsible with its independence with respect to the PKA-negative control".  Figure 1B of their paper that was corrected and published in the Journal of Cell Science [5,145]. Since Greatwall kinase could not phosphorylate S67A-GST-ARPP, the latter should not inhibit GVBD contrary to what is depicted in Figure 1B [5,145]. The results of Figure 1B of the paper by Dupre et al. [143] also contradicted the results published by Mochida [6] and Williams et al [7] if these results can in fact be reproduced.
Mochida [6] and Williams et al [7] stated that dephospho-α-Endosulfine (a homologue of shift of the IC50 to ~700 nM. It is not clear why the authors of paper by Dupre et al. [143] did not find α-Endosulfine in their so called acid and heat stable protein preparation from Xenopus oocytes. The presence of α-Endosulfine in Xenopus oocytes is like sore thumb. In Figure 2A of the paper by Dupre et al. [143], the authors stated that only one single time point (60 min) showed a difference when compared to no progesterone stimulation with p < 0.05. The result is meaningless because one time point difference does not prove anything. The total amount of Arpp19 at the 60 min time point also appeared to be different from no progesterone stimulation. Did the authors determine that there was no significant difference between the total amount of Arpp19 at the 60 min time point and no progesterone stimulation? If not why not? Figure 2C is also problematic because there appeared to be less total Arpp19 at the 30, 60 and 90 min time points when compared with no progesterone stimulation. Again, the authors did not determine whether there were any significant differences between total Arpp19 at the 30, 60 and 90 min times points and no progesterone stimulation. Figure 3A is problematic because it contradicted Figure 1B in that it showed that 775 ng GST-Arpp19 delayed time of GVBD by ~150 min but did not inhibit the extent of GVBD (100 % GVBD was achieved) whereas Figure   1B showed that 775 ng GST-Arpp delayed time of GVBD by ~200 min and inhibited extent of GVBD (only 50% GVBD was achieved). Figure 3A is also problematic because it contradicts the results of Williams et al [7] and Mochida [6] which stated and showed that dephosho-α-Endolfine (a homologue of Arpp19 with same phosphorylation sites) could inhibit PP-2A-B55α and PP-2A-B55ɗ with IC50 of ~500 nM and 1800 nM respectively meaning that 775 ng of GST-Arpp-S109D or GST-Arpp-S67A should both inhibit PP-2A-B55ɗ and trigger resumption of meiosis if the authors' assumption had nothing to do with science fiction.
A second anomaly with the above paper by Dupre et al. [139] is that the authors claimed that they purified acid and heat stable proteins from Xenopus oocytes as described by Boyer et al. [146] but did not show any evidence that they did so. The work of Boyer et al. [146] presented evidence that their acid and heat stable protein preparations consisted of two major proteins of apparent molecular masses 32 kDa and 20 kDa respectively, and that progesterone caused the dephosphorylation of both the 32 kDa and 20 kDa proteins.
What happened to the 31 kDa protein? Immunoblotting an extract of Xenopus occytes with anti-Arpp19 antibodies (See Figure 1 of the paper [143] would also revealed a protein of ~20 kDa. To prove that the acid and heat stable protein of apparent molecular mass ~20 kDa was Arpp19 , the authors should purify and determine part of its sequence, a very easy procedure nowadays. Instead of using recombinant GST-ARPP, the authors should use purified acid and heat stable protein of apparent molecular mass ~20 kDa to show that it prevented GVBD and not GST-Arpp19.
What is perhaps more disturbing about the paper by Dupre et al. [143] is that while the author stated that Arpp19 is the long sought substrate of PKA that is responsible for keeping Xenopus oocytes arrested at prophase and that Dephosphorylation of Arpp19 triggered resumption of meiosis, they do not account for the seminal works of Huchon et al. [147] and Foulkes and Maller [148] which showed that PP-1-I1 and PP-1-I2 would do exactly what Arpp19 phosphorylated on serine 109 by PKA was proposed to do. PP-1-I-1 is a specific inhibitor of PP-1 when it is phosphorylated threonine 34 by PKA [149,150].
PP-1-I-2 is the regulatory subunit of PP-1I which has low basal activity and becomes activated when it is phosphorylated on serine 72 by several activating kinases, including PP-1I.ACK, GSK-3, Cdk1 and Cdk5 [110,111,[151][152][153][154][155][156]. PP-1I could be the enzyme that dephosphorylates serine 287 of Cdc25 causing its activation and triggering resumption of meiosis [115,116] (See below). The inhibition of PP-1I by Inhibitor-1 phosphorylated on threonine 35 by PKA could explain the effect of cAMP and PKA on the inhibition of Cdc25 and Cdk1 and resumption of meiosis. [107,157,158]. The authors of the paper by Dupre et al. [143] do not seem to be able to decide what exactly Arpp19 does in Xenopus oocytes. On the one hand, they claimed that Arpp19 phosphorylated on serine 67 by Greatwall kinase became a specific inhibitor of PP-2A-B55 (although they have not provided any scientific evidence to show that it was so) and that that reaction controlled resumption of meiosis. On the hand, they claimed that Arpp19 phosphorylated on serine 109 by PKA prevented the resumption of meiosis but do not show how. The authors of the paper by Dupre et al. [143] do not know what Arpp19 phosphorylated on serine 109 by PKA would act on, nor do they know whether its dephosphorylation by a protein phosphatase would also trigger the resumption of meiosis (See below). The difference is that the target PP-1-I1 and PP-1-I2, PP-1 has been shown to regulate and dephosphorylate the inhibitory phosphorylation site (serine 287) of Cdc25, the activator of Cdk1 [115][116][117].
In another convoluted scheme of ideas that seemed to have more to do with science fiction than science, the authors of the Dupre et al. [143] proposed that the protein phosphatase that dephosphorylate serine 109 of Arpp19 was PP-2A-B55ɗ, the same protein phosphatase that Arpp phosphorylated on serine 67 by Greatwall kinase was supposed to inhibit in order to trigger resumption of meiosis in Xenopus oocytes. As discussed below, the idea that Arpp19 phosphorylated on serine 109 is phosphorylated by PP-2A-B55ɗ is another fantastical proposal of the authors of Dupre et al. [139]. Greatwall kinase causing Arpp19 to become an inihibitor of PP-2AB55ɗ and that the latter is also the main protein phosphatase that can dephosphorylate Arpp19 at serine 109 that was due to PKA, a chemical reaction that is described as underlying progesterone induced dephosphorylation of Arpp19. Unfortunately for the authors concerned, no verifiable scientific evidence exists that shows that Arpp19 is a specific inhibitor of PP-2AB55ɗ when Arpp19 was phosphorylated at serine 67 by Greatwall kinase and that Arpp-19 phosphorylated at serine 67 and Arpp19 phosphorylated at 109 are both specifically dephosphorylated by PP-2AB55ɗ Arpp19. The idea that Arpp-19 phosphorylated at serine 67 is both an inhibitor and a substrate of PP-2AB55ɗ has more to do with science fiction than science Referring to the papers [1,2], the authors in the paper [9] stated that "PP2A associated with the B55δ subunit appears to be a major enzyme that dephosphorylates Cdk1 targets Extrapolating from the papers described by Mochida et al. and Gharbi-Ayashi et al. [1,2], the authors proclaimed that "Here, we investigate for the first time the detailed role of the Gwl/ARPP19/PP2A module in a physiological cellular process, namely meiotic maturation of Xenopus oocytes. We show that ARPP19 is expressed in prophase-arrested oocytes and is in vivo phosphorylated by Gwl at S67 during meiotic resumption. We reveal that the Gwl/ARPP19/PP2A system plays an essential role during meiotic M-phase entry, not only by counteracting Cdk1 substrate phosphorylation but also by promoting Cdk1 activation. Once activated, it behaves as a major component of the MPF autoamplification loop, rendering this process independent of PKA activity and independent of protein synthesis provided that a small amount Mos is present. Gwl/ARPP19/PP2A therefore not only regulates the events required for M-phase progression and exit, downstream of MPF, but also MPF activation per se and meiotic M-phase entry" [9].
The obvious question that the authors should have asked was why they ruled out other forms of protein phosphatases, including PP-1 and especially PP-1I which is an important form of PP-1. The authors of the paper did not do any experiments that showed that PP-2A-B55ɗ played "an essential role during meiotic M-phase entry, not only by counteracting Cdk1 substrate phosphorylation but also by promoting Cdk1 activation.
Once activated, it behaves as a major component of the MPF auto-amplification loop, rendering this process independent of PKA activity and independent of protein synthesis provided that a small amount Mos is present. Gwl/ARPP19/PP2A therefore not only regulates the events required for M-phase progression and exit, downstream of MPF, but also MPF activation per se and meiotic M-phase entry".
Ruling out PP-1 contradicts the seminal works of Huchon et al. [149] and Foulkes and Maller [150] which showed that a specific inhibitor of PP-1, namely protein phosphatase-1 inhibior-1 (PP-1-I1) or PP-1-I2 delays the resumption of meiosis induced by progesterone but not by MPF in Xenopus oocytes implying the importance of a form of PP-1 in the resumption of meiosis. In view of the fact that the authors in the paper/manuscript [9] did not do any work to rule out the role of any forms of PP-1 in the initiation, maintenance and ending of meiosis in Xenopus oocytes, they cannot scientifically state that no form of PP-1 was not involved. It must be noted that PP-1 exists in many forms and that the activity of PP-1 depends on targeting units [151][152][153] , other modulating proteins including 14-3-3 and whether PP-1 binds directly to its substrates or not [151][152][153][154][155][156][157][158][159][160][161]. A case in point, the PP-1 catalytic subunit binds cdc25 at a RVXM site that is specifically recognized by PP-1 [115,116]. A form of PP-1 termed PP-1 has low basal activity and requires phosphorylation of its regulatory subunit on threonine 72 by PP-1I activating kinase (PP-1I ACK) to exhibit its full activity [110,111,[154][155][156][157][158][159][160]. Both the catalytic subunit and regulatory subunit of PP-1I have been shown to be phosphorylated by Cdk1 [158,161,162]. Further work will show that PP-1I is involved in the direct control of Cdc25. All the evidence currently available supports the view that PP-1I is involved in the direct control of Cdc25 by dephosphorylating serine 287 of Cdc25. PP-1I is complexed to 14-3-3 and C-TAK1 [111] which has been shown to be able to phosphorylate serine 287 of Cdc25 and inhibit it [104] and the regulatory subunit of PP-1I and Cdc25 in a complex of Cdc25-PP-1I-14-3-3-C-TAK1 resulting in the inhibition of both PP-1I and Cdc25 (H.Y.L. Tung, unpublished data). Available scientific evidence also shows that the dissociation of 14-3-3 from Cdc25 can also be effected by phosphorylation of threonine 138 of Cdc25 by Cdk2 and that PP-2A-B56' negatively regulates the dissociation of 14-3-3 from Cdc25 by dephoshorylating threonine 138 of Cdc25 [27]. Thus, the activation of Cdc25 may occur after phosphorylation of threonine 138 and release of 14-3-3 followed by dephoshorylation of serine 287 by PP-1I. Cdc25 is itself phosphorylated on serine 285 and regulated by Cdk1.
It is first important to consider a number of conclusory statements of the authors of the paper/manuscript [9]. It is stated that: (i) Importantly, Cdk1 activation also requires the inhibition of a specific phosphatase, the PP2A-B55ɗ isoform, which counteracts Cdk1dependent phosphorylations of mitotic/meiotic substrates, including Cdc25 and Myt17.
PP2A-B55ɗ inhibition is achieved by Arpp19, a specific inhibitor of this phosphatase when phosphorylated at S67 by the kinase Greatwall (Gwl)". As stated above, that phosphorylation of Arpp19 by Greatwall kinase at serine 67 converts it to a specific inhibitor of PP2A-B55δ is a fallacy that is not based on concrete verifiable scientific data [109]. (ii) "In Xenopus oocyte, it is clearly established that S67 phosphorylation of Arpp19 by Gwl promotes its binding to PP2A-B55δ and the inhibition of the phosphatase". As stated above, that phosphorylation of Arpp19 by Greatwall kinase at serine 67 converts it to a specific inhibitor of PP2A-B55δ is a fallacy that is not based on concrete scientific data [109,145]. (iii) "The molecular regulation of this last step, involving Gwl, Arpp19 and PP2A, has been well deciphered in mitosis and meiosis". "In Xenopus oocyte, it is clearly established that S67 phosphorylation of Arpp19 by Gwl promotes its binding to PP2A-B55δ and the inhibition of the phosphatase". As stated above, the role of Arpp19 phosphorylated by Gwl at serine 67 as a specific inhibitor of PP-2A B55δ is a fallacy that is not based on concrete scientific data [109,145]. (iii) "we identify PP2A-B55ɗ as the phosphatase that dephosphorylates Arpp19 at S109, thus enabling oocytes to resume meiosis. The level of Arpp19 phosphorylated at S109 in prophase-arrested oocytes results from a balance between PKA and PP2A-B55ɗ activities in favor of the kinase. Upon hormonal stimulation, PP2A-B55δ activity remains unchanged while PKA is downregulated, leading to the partial dephosphorylation of Arpp19 at S109 that unlocks the prophase arrest. Therefore, the timing of meiosis resumption relies on the temporal coordination of S109 and S67 phosphorylations of Arpp19, orchestrated by one single phosphatase, PP2A-B55ɗ, opposing two kinases, PKA and Gwl". The authors have not provided any shred of scientific evidence that proved that "PP2A-B55ɗ was the protein phosphatase that dephosphorylated serine109 of Arpp19, that the dephosphorylation reaction was connected to resumption of meiosis, that the level of Arpp19 phosphorylated at S109 in prophase-arrested oocytes results from a balance between PKA and PP2A-B55ɗ activities in favor of the kinase, that upon hormonal stimulation, PP2A-B55δ activity remains unchanged while PKA is downregulated, leading to the partial dephosphorylation of Arpp19 at S109 that unlocks the prophase arrest and that the timing of meiosis resumption relies on the temporal coordination of S109 and S67 phosphorylations of Arpp19, orchestrated by one single phosphatase, PP2A-B55ɗ, opposing two kinases, PKA and Gwl".
The authors of the paper [9] proclaimed that they identified PP-2AB55ɗ as the protein phosphatase that is responsible for dephosphorylating serine 109 of ARPP19. The authors used GST-tagged Arpp19 previously in vitro phosphorylated at S109 by PKA (pS109-GST-Arpp19) as substrate to assay for protein phosphatase(s). In Figure 1 of the paper [9], the protein phosphatase that acted on pS109-GST-Arpp19 was determined by Western Immunoblotting of pS109-GST-Arpp19 following incubation with extracts of prophase Xenopus oocytes in the presence of 1 or 10 µM okadaic acid. The authors observed dephosphorylation of pS109-GST-Arpp19. However, 1 or 10 µM okadaic acid would not allow them to determine the nature of the protein phosphatase. It is note worthy that according to Figure 1C, almost ~30 % of total protein phosphatase activity was not inhibited by 10 µM okadaic acid, assuming that the authors knew how to assay for total protein phosphatase activity. A large proportion of PP-1 is in the form of PP-1I which is inhibited by high concentration of ATP and requires Mg 2+ and low concentration of ATP to exhibit full activity [110,111,[151][152][153][154][155][156] and a large proportion of PP-2A is in its latent form and requires activating molecules to exhibit full activity [36,121]. The result of Figure 1C showed that ~30% of total protein phosphatase activity was not due to PP-1 and PP-2A, and that the amount of total protein phosphatase activity being measured was incorrect and misleading.
In Figure 2, the authors of the paper [9] used ammonium sulfate precipitation to apparently separate PP-1 and PP-2 activities. The result of Figure 2 was meant to show that PP-2A could be recovered with 40% ammonium sulfate precipitation which contained PP-2A and a small amount of PP-1 was the predominant protein phosphatase that acted upon pS109-GST-Arpp19. This is a misleading statement since the specific activity of PP-1 is an order of magnitude higher than PP-2A. This is basic enzymology.
The fact that one sees less protein does not mean that its activity is lower. The activity of the enzyme depends on its specific activity not on the amount of protein one sees.
In Figure 3B, the authors of the paper [9] used Mono Q column to separate the protein phoshatase activities that acted upon pS109-GST-Arpp19 and stated that PP-2A-B55ɗ was the main protein phosphatase that acted upon pS109-GST-Arpp19. Unfortunately, the statement is misleading because: (i) the peak of activity does not coincide with the Since the Flow Through Fractions were not analyzed and were discarded, PP-1 would not be observed in the subsequent Superose 10 Gel Filtration Chromatorgraphy.
In Figure 5, the authors of the paper [9]  To then come up with a scheme that depicts Arpp19 as both an inhibitor and a substrate of PP-2A-B55ɗ requires a lot of imagination and mental acrobatics that has more to do with science fiction than science. The first part of the scheme depicted in Figure 9  Arpp67 is itself riddled with anomalies and unscientific data [7]. As discussed in [109], the paper in question did not show that that PP-2A-B55α was the protein phosphatase that dephosphorylated serine 67 of Arpp19.
In the second part of the scheme, the authors of the paper [9] showed that upon stimulation with progesterone, PKA is inhibited, resulting in dephosphorylation of serine In the third part of the scheme, the authors of the paper stated that Greatwall kinase phosphorylates Arpp19 at serine 67 converting it to a specific inhibitor of PP-2A-B55ɗ.
As stated above, the evidence that showed that Arpp19 phosphorylated at serine 67 was a specific inhibitor of PP-2A-B55ɗ is misleading at best and dishonest at worse. In order to show that Arpp19 phosphorylated at serine 67 is a specific inhibitor of PP [149] which showed that PP-1-I1 which becomes a specific inhibitor of PP-1 when it is phosphorylated on threonine 35 by PKA interferes with Progesterone effect in Xenopus oocytes arrested at prophase and the paper by Foulkes and Maller [150] which showed that PP-1-I2 delays the effect of progesterone in Xenopus oocytes arrested at prophase, nor does the scheme reconciled itself with the work of Duckworth et al. [107] which showed that (i) Cdc25, the activator of Cdk1 is itself phosphorylated by PKA on serine 287 which inhibited Cdc25, (ii) serine 287 is dephosphorylated in response to progesterone just before cdc2 dephosphorylation and Mphase entry, (iii) high PKA activity maintains phosphorylation of Ser-287 in vivo, and (iv) inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287, and the work of Margolis et al. [115,116] which showed that (i) PP-1 was required to dephosphorylate serine 287 of cdc25, (ii) the effect of PP-1 on cdc25 was antagonized by PP-1-I1, (iii) PP-1 forms a complex with cdc25, and (iv) and that dephosphorylation of serine 287 of cdc25 is masked by 14-3-3 during interphase, Why the authors of the paper [9]  Arpp19 to a better substrate for Greatwall kinase?. These are the questions that must be answered if a role for the dephosphorylation of serine 109 of Arpp19 is to be found. These kind of studies will require proper Enzyme Chemistry and Enzyme Kinetics works (tasks that the authors of the paper/manuscript did not seem to know about) and not Western Immunoblotting type work. Mochida [6] showed and stated that phosphorylation of α-Endolfine on serine 109 by PKA antagonized the effect of α-Endolfine phosphorylated on threonine 28 by Cdk1 on PP-2A-B55ɗ by shifting the IC50 from 130 nM to 700 nM (if the results are to be believed as the author of the paper [6] did not seem to know how to perform a protein phosphatase inhibitory assay). If the effect of the phosphorylation of α-Endolfine on serine 109 by PKA can be repeated with Arpp19, it follows that the function of Arpp19 phosphorylated on serine 109 by PKA would only serve to antagonize the inhibition of PP-2A-B55ɗ by Arpp phosphorylated on threonine 28 by Cdk1 and not to keep Xenopus oocytes arrested at prophase as suggested in [5,9,143].
The results described by Dupre et al. [5], Lemmonier et al. [9] and Dupre et al. [143] are at variance with those of Hara et al. [163] which showed that "Greatwall kinase is not B55ɗ by a factor of at least 4 and that only `50% inhibition could be observed, it would imply that the effects thiophospho-Arpp19 were most probably substrate directed.
However, since no information with respect to the amount and concentration of the substrate was provided, it is impossible to tell whether the effects of thiophospho-Arpp19 was enzyme or substrate directed or both. This would be significant because, if the effects of hiophospho-Arpp19 was substrate directed, it would imply that there would be virtually no inhibition of PP-2A-B55ɗ in the Star fish oocytes. Second, the authors, Okumura et al. [8] simply stated in the Method Section that PP-2A-B55ɗ was assayed for its ability to dephosphorylate 32 P-labeled Fizzy-pSer50 (5000 cpm) in the presence of thiophosho-Arpp19 phosphorylated by Cdk1 but did not state what the specific radioactivity of the substrate was. Could the authors have accurately measure less than 500 cpm of 32 P-labeled phosphate released in the control and less than 250 cpm 32 Plabeled phosphate released in the experiment in which thiophospho-Arpp was added. (in order to be in the linear part of the enzyme reaction, it is important to assay less than 10% dephosphorylation of the substrate). As stated above, in order to show that Arpp19 The results described by Dupre et al. [5], Lemmonier et al. [9] and Dupre et al. [143] are in stark contradiction with the work of Adhikari et al. [164] which showed that (i) Mastl kinase (equivalent of Greatwall kinase) was not required for the resumption of meiosis and progression to metaphase I in mouse oocytes, (ii) Mastl-deficient ooctes did not fail to porgress through meiosis I but underwent GVBD with kinetics and efficiencies that were indistinguishable from those of Mastl +/+ oocytes, (iii) Mastl has no essential role during meiotic resumption or prometaphase I progression in mouse oocytes, (iii) there might be some slight differences in the timing of completion of meiosis I between control and mutant oocytes in vivo, (iv) Mastl was required for the timely activation of APC/C that was needed for the completion of meiosis I in mouse oocytes, (v) The activation of spindle assembly checkpoint occurred normally in Mastl deficient oocytes, (vi) The level of Cdc20 in Matl deficient oocytes remained low compared to normal oocytes, (vii) Mastl was essential for MII entry, (viii) Mastl was not required for the upregulation of Cdck1 activity during prometaphase I but was required for entry in MII, (ix) PP-2A activity in both Mastl deficient and normal oocytes were comparable at GV and GVBD stages, implying that mouse oocytes progress through metaphase I without the suppression of PP-2A activity and that Mastl did not regulate PP-2A activity during prometaphase I, and (x) Mastl likely play an essential role in triggering the activation of APC/C that is required for the down regulation of Cdk1 activity that might mediate the timely onset of anaphase I in mouse oocytes.
To summarize, the papers by Dupre et al. [5], Lemmonier et al. [9] and Dupre et al. [143] are just copycat papers that hinge on the misplaced assumption that Arpp19 is a specific inhibitor of PP-2A-B55ɗ when the latter is phosphorylated by Greatwall kinase (recently reviewed in [109,147]. Now, the authors of the paper [9] make further statements that are at best misleading and at worse dishonest as they have provided no scientific verifiable evidence that PP-2A-B55ɗ is the protein phosphatase that dephosphorylates both serine 67 and serine 107 of Arpp19. The authors of the papers by Dupre et al. [5], Lemmonier et al. [9] and Dupre et al. [139] do not seem to be able to decide what exactly Arpp19 does in Xenopus oocytes. On the one hand, they claimed that Arpp19 phosphorylated on serine 67 by Greatwall kinase became a specific inhibitor of PP-2A-B55 (although they have not provided any scientific evidence to show that it was so) and that that reaction controlled resumption progression of meiosis. On the other hand, they claimed that Arpp19 phosphorylated on serine 109 by PKA prevented the resumption of meiosis but did not state how. The authors of the Dupre et al. [5], Lemmonier et al. [9] and Dupre et al. [139] completely ignored published works that contradicted their work and did not provide any reasons to the readers and Scientific Researchers who are trying to decipher who is/are telling the truth and whether to spend resources in man hours to try to repeat their published works that are at best misleading and at worse dishonest. The idea that a form of PP-2A could undergo marked inhibition during resumption of meiosis is not compatible with the known function of PP-2A as a key controller of cell transformation and as tumor suppressor [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100].
The role of PP-2A in the control of meiosis.
The following is a more realistic model for the role of PP-2A in the control of meiosis.