Dynamic Equilibrium of Protein Phosphorylation by Kinases and Phosphatases Visualized by Phos-Tag SDS-PAGE
, Kento Nishikawa 2, Kento Hiraishi 2, Kaku Shimoji 2, Kenichi Nagase 1 and Eiji Kinoshita 3
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript effectively utilizes Phos-tag SDS-PAGE to visualize the roles of kinases and phosphatases in cellular phosphorylation events. By focusing on 20 intracellular proteins in HeLa cells, it provides valuable insights into the maintenance of phosphorylation equilibrium by serine/threonine and tyrosine phosphatases. The study's findings suggest that many kinase-mediated phosphorylation events are incidental, underscoring the importance of phosphatases in regulating these modifications to preserve cellular function. This work broadens the understanding of phosphorylation dynamics, highlighting the extensive and often overlooked role of phosphatases.The manuscript is well-written, and the data are presented clearly. While the references are appropriate, the manuscript would benefit from including more recent studies. This would provide a more up-to-date context for the research and highlight its relevance in light of current findings.
Author Response
reviewer's comment
The manuscript effectively utilizes Phos-tag SDS-PAGE to visualize the roles of kinases and phosphatases in cellular phosphorylation events. By focusing on 20 intracellular proteins in HeLa cells, it provides valuable insights into the maintenance of phosphorylation equilibrium by serine/threonine and tyrosine phosphatases. The study's findings suggest that many kinase-mediated phosphorylation events are incidental, underscoring the importance of phosphatases in regulating these modifications to preserve cellular function. This work broadens the understanding of phosphorylation dynamics, highlighting the extensive and often overlooked role of phosphatases.The manuscript is well-written, and the data are presented clearly. While the references are appropriate, the manuscript would benefit from including more recent studies. This would provide a more up-to-date context for the research and highlight its relevance in light of current findings.
our response
Your constructive comments are greatly appreciated. We have replaced references 2-4 in the Introduction section with more recent ones. Page13 line 501-505.
We have not replaced older references if they were the source of important findings.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have almost twenty years of experience with the Phos-tag system, on which their first report was published in 2006 and for which also a company has been established. Since then, they have published about 50 original or review articles on this topic, describing various applications of this technology. In this manuscript, the authors have evaluated the effects of Ser/Thr or Tyr phosphatase inhibitors on the phosphorylation states of 20 substrates of Ser/Thr or Tyr kinases. They had previously observed hyperphosphorylation of several abundant cytoskeletal proteins by treatment with the Ser/Thr phosphatase inhibitor calyculin A (ref. 5), while now they have extended these studies to include also the Tyr phosphatase inhibitor pervanadate and have been able to detect changes also in less abundant signaling proteins, such as transcription factors and kinases by combining Phos-tag SDS-PAGE to immunoblotting. Technically the data are sound and fit well with the scope of the journal, but the conclusions based on them are not as convincing.
Major points:
- The structure of the Results section is unsually repetitive, with short descriptions of each substrate and its phosphorylation state after treatments. The section also contains a lot of speculation that would better fit to Discussion, e.g. suggestions on how certain substrates are “constantly and routinely phosphorylated by multiple Ser/Thr and/or Tyr kinases”
- How specific are the antibodies, so how likely it is that the bands indicated by arrows or arrowheads really represent the proteins of interest?
- The significance of the data remains unclear, as also the authors state that “protein phosphorylation in the presence of phosphatase inhibitors is random and incidental with no biological significance due to the disordered kinase reaction with loss of reversibility”
- Some other means should be used to confirm that the hyperphosphorylated bands are real and not just treatment artefacts that would not be seen under normal physiological conditions.
Minor points:
- Fig. 1: unclear which lanes are which. C refers to control lane, but presumably calyculin A is in lane 1 (not 2) and pervanadate in lane 2 (not 3)?
Comments on the Quality of English Language
The quality of English language is mostly fine, but some minor corrections are needed.
Author Response
Major points:
comment 1
- The structure of the Results section is unsually repetitive, with short descriptions of each substrate and its phosphorylation state after treatments. The section also contains a lot of speculation that would better fit to Discussion, e.g. suggestions on how certain substrates are “constantly and routinely phosphorylated by multiple Ser/Thr and/or Tyr kinases”
Our response to comment 1
Your constructive comments are greatly appreciated.
The results section is unusually repetitive, but an independent description of each blot is required, and no alternative method of description is found. Phrases containing speculation about the mode of phosphorylation, i.e. words such as "randomly", "constantly", "routinely", "incidentally" and "on a routine basis" have been removed from the Results section. These sentences are marked in red in the revised manuscript.
page4 line 109,115,121,125,131,133,143
page5 line 147,162,170,180
page7 line 238,246,247,255,262
page8 line 285, 289, 298, 305
page9 line 328,336,344,346
page10 line 357, 365,374
comment 2 and 4
2. How specific are the antibodies, so how likely it is that the bands indicated by arrows or arrowheads really represent the proteins of interest?
4. Some other means should be used to confirm that the hyperphosphorylated bands are real and not just treatment artefacts that would not be seen under normal physiological conditions.
Our response to comment 2 and 4
Phos-tag SDS-PAGE assumes that all detected bands are the protein of interest. Therefore, we have confirmed that a band with a high signal-to-noise ratio that is completely specific for the protein of interest is confirmed by conventional SDS-PAGE. As described in section 2.1 of the Results section, the specificity of different types of antibodies in immunoblotting was investigated and 20 antibodies were selected that were sufficiently specific and had high signal-to-noise ratios.As supplemental data, we provide general SDS-PAGE immunoblotting data of 20 different antibodies used in this study. The supplemental data will prove that the bands indicated by the arrows are the phosphorylated species of the protein of interest and not just artifacts.
Comment 3
The significance of the data remains unclear, as also the authors state that “protein phosphorylation in the presence of phosphatase inhibitors is random and incidental with no biological significance due to the disordered kinase reaction with loss of reversibility”
Our response to comment 3
Regarding the first paragraph of the Discussion section.
From the results of this study, it is reasonable to assume that "Protein phosphorylation in the presence of these phosphatase inhibitors would be random and incidental with no biological significance due to the disordered kinase reaction with loss of reversibility." However, this seemed to be an abrupt statement with poor sentence cohesion and a leap in thought. Therefore, the following sentence was added before this sentence. Here we used the words "constantly" and "routinely", which were removed in the Results section.
“All immunoblots showed a Phos-tag pattern indicating increased phosphorylation by one or both phosphatase inhibitors. This suggests that Ser/Thr and Tyr kinases constantly phosphorylate various proteins on a routine basis.”
page10 line 392-294
Minor points:
comment 1
- Fig. 1: unclear which lanes are which. C refers to control lane, but presumably calyculin A is in lane 1 (not 2) and pervanadate in lane 2 (not 3)?
Our response to comment 1
The footnotes in the figure were corrected to calyculinA(lane 1), pervanadate(lane 2).
page 6 line 195-196
page8 line 265-266
page9 line 309-310
page10 line 379
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI was satisfied with most of the changes made by the authors. However, I would not state that "All immunoblots showed a Phos-tag pattern indicating increased phosphorylation by one or both phosphatase inhibitors" as the inhibitors do not phosphorylate anything, but phosphorylation of the proteins is increased in the presence of them.
I am also still concerned about the specificity of the bands. Even though the antibodies used are highly specific, as demonstrated in the suppl. figure 1, there are additional bands present in the Phos-tag control samples, so in case there is no other way to confirm the specificity in Phos-tag SDS Page, this should at least be explained or discussed.
Comments on the Quality of English LanguageMinor corrections needed, but otherwise fine.
Author Response
Your constructive comments are greatly appreciated. Below are our responses to the comments.
comment 1
I was satisfied with most of the changes made by the authors. However, I would not state that "All immunoblots showed a Phos-tag pattern indicating increased phosphorylation by one or both phosphatase inhibitors" as the inhibitors do not phosphorylate anything, but phosphorylation of the proteins is increased in the presence of them.
our response to comment 1
As you pointed out, the inhibitor did not actively phosphorylate the protein, but the protein was phosphorylated by the kinase in the presence of the inhibitor. Therefore, "by" has been replaced by "in the presence of" in the Abstract and Discussion sections.
Page1 line 15
Page10 line 394
comment 2
I am also still concerned about the specificity of the bands. Even though the antibodies used are highly specific, as demonstrated in the suppl. figure 1, there are additional bands present in the Phos-tag control samples, so in case there is no other way to confirm the specificity in Phos-tag SDS Page, this should at least be explained or discussed.
our response to comment 2
In the normal SDS-PAGE and immunoblotting shown in Figure S1, almost no bands other than the specific band are detected.
As you pointed out, additional bands were detected in the control sample of the Phos-tag SDS-PAGE.
This is because there are multiple phosphorylated species in steady-state cells. This was noted in the description of most blots in the Results section as follows
We forgot to mention the addition of FigS1 in the previous revised manuscript, so we have included it in this revised version.
page 2 line 85
