Peer review of: Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

PP1 and PP2A are major cellular S/T phosphatases and understanding of their substarte selectivities 
is a fundamentally important question relevant to all biomedical fields. The study by Köhn laboratory 
addresse this question by phosphoproteome analysis of both peptide libraries and cellular lysates 
treated transiently with recombinant PP1 and PP2A catalytic subunits (PP1c and PP2Ac, 
respectively). 

 

Although some of the main conclusions have been suggested by the previous studies, this work 
provides very important unbiased information to complement our understanding of PP1 and PP2A 
intrinsic substrate preferences. Authors have performed extensive quality control experiments to 
minimize the likelihood that the observed results are not due to technical bias. However, many 
results requre statistical significane analysis which would be essential to evaluate the significance of 
the findings, but could also help the authors to further squeeze out from their data the most reliable 
and meaningful conclusions. 

 

General comments: 

 

1. As this study potentially fills previous knowledge gaps and substantiate the previous findings 
related to substrate peptide selectivity of PP1c and PP2Ac, it would be very important to clearly 
discuss what is the advance from this study, as compared to previous studies, and what would be 
the current master model when putting all existing studies together. In addition to discussing in 
more detail the model in light of their own data and already referenced papers from Saurin, Nilsson 
and Peti laboratories, they should also include other recent important studies such as the study by 
Hendus-Altenburger et al., which addressed the importance of flanking aminoacids for Calcineurin-
mediated dephosphorylation (Nat Commun. 2019 Aug 2;10(1):3489). 

 

 

2. It would be very important that authors even more clearly indicate, starting from title, abstract 
and introduction, that at least currently there are no examples of dephosphorylation of S/T by 
catalytic subunits alone. Rather the presented results presented are relevant for understanding 
which S/T residues could be preferentially dephosphorylated by PP1 and PP2A, AFTER they have 
been recruited to the target/target complex by regulatory subunits. Thereby the authors could 
prevent misunderstanding that they are proposing a new mode of function for PP1c and PP2Ac as 

monomers. Along these lines, starting from the title, I would not talk about PP1 and PP2A substrate 
specificity but rather about aminoacid sequence determinants for dephosphorylation by catalytic 
subunits of PP1 and PPP2A 

 

3. Please explain clealry in the text that even though catalytic subunits have T/S preference, still vast 
majority of all dephosphorylated sites in our cells are serines. Otherwise the reader might leave with 
wrong perception that majority of dephosphorylation events by these PPases occur on Threonines. 

 

Comments to data and figures 

 

4. To ensure the purity of proteins, please provide full gel coomassie staining images of purified PP1c 
and PP2Ac used in the experiments. 

 

 

5. Figure 1: What is the basis for using AAAA streches before and after the phosphorylated S/T? How 
is it controlled that this not affect the results as no such motifs are found in nature in most target 
sites. 

 

6. Fig. 2B: The patterns for PP1 and PP2A look pretty similar. Which aminoacid preferences are 
statistically significantly different between PP1 and PP2A? 

 

7. Fig. 3A): 

-Doesn´t CalA in the lysate inhibito also the recombinant PP1c and PP2Ac? 

- What is the approximate molar ratio between recombinant PPase and substrate proteins in the 
lysate 

 

8. ln. 240: This is a very important potential caveat of the study. Please demonstarte the degree of 
incorporation of recombinant PP1c and PP2Ac to holoenzyme complexes by gel-filtration analysis 
and affinity purification coupled with MS. 

 

9. 3D) Are these enrichments statistically significant? 

 

10. 3E) Similar to 2B, which aminoacid preferences are statistically significantly different between 
PP1 and PP2A? Even more importantly, after calculating the significances for both 2B and 3E, what 
are the aminoacid statistically significant aminoacid preferences that are consistent between both 
methods (FIg. 2 and 3). This would be very important and clear take a home message from the paper 
with statistical power. 

 

11. Fig. 4: It is unclear whether this analysis is based on data from Fig. 2 or Fig. 3? Again, related to 
comments above, similar analysis should be done with both datasets. 

 

12. Ln. 276. Is the authors final conclusion that after all there is no aminoacid preference for 
PP2Ac?? This is very confusing as related to overall message of the paper. Please clarify. 

 

13. Ln. 281-283: The alternative explanation would be that as PP1 functions a s dimer it more easily 
assembles to holoenzyme than PP2A in these in vitro lysate conditions and therefore its shows more 
strict substrate selectivity. This further indicates that authors need to provide the experimental 
evidence requested in my comment 8. 

 

14. Fig. 4C; ln. 286-291: Are these enrichments statistcially significant between PP1 and PP2A. 

 

15. Ln. 38: I would prefer that the authors would not strengthen the misconception of the inbalance 
of kinase and phosphatases. Human genome has 40 genes that code for the catalytic subunit of S/T 
PPases, but at least as many functional PPases (as functional complexes) as there are kinases. 

 

16. Ln. 180-181: ”that the close proximity of Pro to the pSer/Thr could have suggested an active-site-
mediated recognition of the SP motif” For a non-expert reader this is hard to understand 

 

17. ln. 259: ”class I sites (localization probability of phosphorylation >0.75” For a non-expert reader 
this is hard to understand 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The authors in the manuscript entitled “Dissecting the basic substrate specificity of phosphoprotein 
phosphatases 1 and 2A through phosphoproteomic approaches” have explored large 
phosphopeptide library to show the pThr specificity of PP1 and PP2 phosphatases. The generation 
and validation of a reliable phosphopeptide library is crucial for this study. Therefore, authors have 
purified and verified great deal of different phosphopeptides to carry out PP1 an PP2A 
dephosphorylation assays. The biggest flaw effecting the whole study is the fact that only 
recombinant catalytic subunits (PPP1CA & PPP2CA) where used in the dephosphorylation assays. 
PP1 and PP2A, as well as other protein phosphatases form large protein complexes with multiple 
regulatory subunits regulating their activities (for example PMID: 27880917 and PMID: 28330616). 
The main open question is how and if the substrate specificity/preference changes when the PP1 
and PP2 regulatory subunit(s) are also used in the assays? 

Nevertheless, the authors also tested their conclusion by in vivo (HeLa cell line) phosphoproteomics 
analysis, where both PP1 and PP2 again showed intrinsic preference for pThr sites. They have also 
investigated if this preference is somehow affected by the any presence of other amino acids around 
the PThr/pSer. In this quest they have found RXXpS motif, in several PP1 and PP2 substrates as well 
as in 14-3- binding site. One of the identified substrates is GAB2 which also bind with 14-3-3 binding 
site containing proteins (14-3-3 beta) have been dephosphorylated by PP1 in vitro, leading to 
disruption of GAB2-14-3-3 interaction. This extent of dephosphorylation was also monitored by MS 
using label-free quantification, where they found PP1 specificity towards RXXpS motif. 

 

 

Major criticism: 

 

The authors should verify how the presence of the regulatory subunit(s) affect PP1 and PP2A 
substrate specificity. 

 

 

Minor comments: 

 

Only one cell line was used in vivo assay. The use of (an)other cell line(s) would strengthen the 
manuscript. Additionally for authentication of the cell line statement “to the best of our knowledge 
these are HeLa cells” is not sufficient. See for example a great study on the HeLa heterogeneity by 
the Aebersold group “PMID: 30778230” 

 

Minor corrections 

 

-Page 19 , line 10 ----(Fig 6a) should be Fig 5c, 

line 11------Fig 6b should be Fig 5d, 

line 12-------(Fig 6c) should be Fig 5e, 

line 14-------(Fig 5b) should be Fig 5c. 

 

-Figure 5a should show error bars. In its legends, e, should be legend d and f, should be legend e.