β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-Releasing Hormone and Taltirelin

Round 1

Reviewer 1 Report

In the paper entitled “β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-releasing Hormone and Taltirelin” the authors studied phosphoproteome of normal and β-arrestin2-silenced pituitary GH1 cells after 30 min treatment with TRH or Taltirelin by using nLC-MS2 method and relevant data analysis with the aim to point the importance of b-arrestin 2 for TRH receptor agonists action as well to show differences between TRH and Talterin mediated intracellular signaling.  By usage of specific software’s and bioinformatics databases the authors pointed to some differences in signal transduction associated with small GTPases and MAP kinases between experimental groups. The provided results possess novel aspect since up to now there is no published data on TRH or its analog action in respect to beta-arrestin2-dependency at the level of detailed analysis of phosphosignaling pathways. The paper could be published, however before it needs extensive revision and clarification of some issues as listed below:

1. Title suggest “critical involvement of β-Arrestin2 in the differential regulation …” however it should be noticed that beta-arrestin2 silencing was not complete (Fig. S1). Could this observation somehow affect performed by authors data interpretation? Moreover for gene silencing of beta-arrestin 2 only one siRNA vector was used (should be at least 2) and there is no verification of its specificity to beta-arrestin2 (there should be verification if this siRNA does not affect for example other members of arrestin family). Some discussion in this respect should be done. Moreover, for Fig. S1 there should be presented protein load control and data should be calculated to estimate the percentage of gene silencing.  
2. In Introduction part should be some information about types of TRH receptors present I rodents and differences between them. And consequently in Discussion part should be some information if studied signaling pathways refers only to TRHR1 receptor? I expect that in pituitary cells this type of receptor predominates. However it should be also underlined that in respect to potential neuroprotective effects of TRH or its analogs rather TRHR2 is thought to be involved. It should be somehow included in the paper.
3. The results section is too detailed and is too long. I would rather relocate this very detailed description in Supplementary data (including also relevant References to this section) and in main Text leave the most significant results which differentiate the studied experimental groups. In this way Reference list (now 252 papers) could be shortened.
4. Results description and discussion of data may be done together – in fact in present chapter Discussion is done in this way. This could be a little bit extended as well limitations of the study should be included.

Author Response

Response to the Reviewers´ comments

We would like to thank the reviewers for their insightful and very thorough comments on the manuscript. We have revised the manuscript accordingly. We have modified the manuscript according to the reviewers´ suggestions as much as possible. Below is our response to the issues raised in the review.

 

Reviewer 1

In the paper entitled “β-Arrestin2 Is Critically Involved in the Differential Regulation of Phosphosignaling Pathways by Thyrotropin-releasing Hormone and Taltirelin” the authors studied phosphoproteome of normal and β-arrestin2-silenced pituitary GH1 cells after 30 min treatment with TRH or Taltirelin by using nLC-MS2 method and relevant data analysis with the aim to point the importance of b-arrestin 2 for TRH receptor agonists action as well to show differences between TRH and Talterin mediated intracellular signaling.  By usage of specific software’s and bioinformatics databases the authors pointed to some differences in signal transduction associated with small GTPases and MAP kinases between experimental groups. The provided results possess novel aspect since up to now there is no published data on TRH or its analog action in respect to beta-arrestin2-dependency at the level of detailed analysis of phosphosignaling pathways. The paper could be published, however before it needs extensive revision and clarification of some issues as listed below:

1. Title suggest “critical involvement of β-Arrestin2 in the differential regulation …” however it should be noticed that beta-arrestin2 silencing was not complete (Fig. S1). Could this observation somehow affect performed by authors data interpretation? Moreover for gene silencing of beta-arrestin 2 only one siRNA vector was used (should be at least 2) and there is no verification of its specificity to beta-arrestin2 (there should be verification if this siRNA does not affect for example other members of arrestin family). Some discussion in this respect should be done. Moreover, for Fig. S1 there should be presented protein load control and data should be calculated to estimate the percentage of gene silencing.

Response: We agree with the Reviewer comment that beta-arrestin2 silencing was not complete. Indeed, beta-arrestin2 expression was reduced by more than 60 % in cells trensfected with beta-arrestin 2 siRNA. In parallel, there was no change in beta-arrestin1 expression. This information was added to Supplementary Fig. S1, including the loading control to estimate the percentage of gene silencing. In the Discussion section of the revised manuscript, we added incomplete β-arrestin2 silencing as one of the limitations of the present study and explained that differential reduction of β-arrestin2 expression could potentially affect cell signaling and biological processes to different extents. Nevertheless, we believe that even the incomplete silencing of beta-arrestin2 provides clear evidence for its crucial role in the differential regulation of phosphosignaling pathways by TRH and TAL.

 

2. In Introduction part should be some information about types of TRH receptors present I rodents and differences between them. And consequently in Discussion part should be some information if studied signaling pathways refers only to TRHR1 receptor? I expect that in pituitary cells this type of receptor predominates. However it should be also underlined that in respect to potential neuroprotective effects of TRH or its analogs rather TRHR2 is thought to be involved. It should be somehow included in the paper.

Response: We greatly appreciate this helpful comment by the reviewer. We have added some information about the subtypes of TRH-R in the Introduction section of the revised manuscript. And we have also included some related comments in the Discussion section. We believe that the neuroprotective effects of TRH may be mediated, at least in part, by TRH-R1. In humans, a TRH-R subtype is expressed that is structurally homologous to TRH-R1. Moreover, the CNS effects of taltirelin in mice have been shown to be mediated primarily, if not exclusively, by TRH-R1 (Thirunarayanan, 2013, Neuropsychopharmacology, 38, 950-956). If the neuroprotective effects of TRH and taltirelin were mediated only by TRH-R2, then no neuroprotective effects would be observed in human SH-SY5Y cells (Jaworska-Feil, 2010, Neuropeptides. 44(6): 495; Zheng, 2018, Front Cell Neurosci. 12: 485).  

 

3. The results section is too detailed and is too long. I would rather relocate this very detailed description in Supplementary data (including also relevant References to this section) and in main Text leave the most significant results which differentiate the studied experimental groups. In this way Reference list (now 252 papers) could be shortened.

Response: Following the Reviewer recommendation, we have moved some descriptive results to the Supplementary Results to shorten the Results section. However, this has little effect on the length of the bibliography. More than ninety references are cited exclusively in the figure legends. Many references, mentioned in the text of the results, are also cited in the figure legends. The only way to shorten the bibliography would be to move the references cited in the figure legends to the Supplementary References. However, we fear that this would not be a correct citation method and therefore prefer to leave most references in a standard reference section of the manuscript.

 

4. Results description and discussion of data may be done together – in fact in present chapter Discussion is done in this way. This could be a little bit extended as well limitations of the study should be included.

Response: We disagree with the Reviewer suggestion to combine the Results and Discussion sections. In the Results section, the results for each signaling pathway are described in detail. In the Discussion section, we have attempted to link the results from the different signaling pathways to draw broader conclusions. Consistent with the Reviewer´s suggestion, we have added limitations to the study at the end of the Discussion section of the revised manuscript.

Reviewer 2 Report

The authors did and important study to dissect the distinctions in Phospho-signaling pathways activated by TRH & Taltirelin. It also successfully shows the distinct pathways activation is due to biased agonism, as suggested by the phosphorylation profiles in β-arrestin2 Knockdown cells.

Despite the extensive phospho proteomics study this manuscript needs some more data to drive home the conclusions drawn for the study.

The data represented as different signaling pathways reflecting changes in conditions of Arr/C, TRH/C, TAL/C, Arr-TRH/C, Arr-TAL/C is a good attempt but difficult to follow.

The supplementary data corresponding to each figure contains fold changes value in square brackets for some Phosphosites. It would be straightforward for the readers to understand if the data is represented as graphs, which would be just a fold representation of the phosphoproteins varying. The authors have Graphpad PRISM software which would this job further easy.

In addition, we suggest the authors to highlight the phosphoproteins which show significant variations in  each pathway either positive or negative way.

It would have better if the if the supplementary table was available as excel sheet.

Author Response

Response to the Reviewers´ comments

We would like to thank the reviewers for their insightful and very thorough comments on the manuscript. We have revised the manuscript accordingly. We have modified the manuscript according to the reviewers´ suggestions as much as possible. Below is our response to the issues raised in the review.

 

Reviewer 2

The authors did and important study to dissect the distinctions in Phospho-signaling pathways activated by TRH & Taltirelin. It also successfully shows the distinct pathways activation is due to biased agonism, as suggested by the phosphorylation profiles in β-arrestin2 Knockdown cells.

Despite the extensive phospho proteomics study this manuscript needs some more data to drive home the conclusions drawn for the study.

The data represented as different signaling pathways reflecting changes in conditions of Arr/C, TRH/C, TAL/C, Arr-TRH/C, Arr-TAL/C is a good attempt but difficult to follow.

Response: We are very pleased with the reviewer´s positive evaluation of our work. This study is very complex, as it contains a large amount of data and may seem overloaded. We believe that understanding all aspects of this study will take a lot of time and that readers will need to be familiar with several topics, such as protein relationships in signaling pathways, protein structure and sequences, or ligand disposition to biased agonism. 

The supplementary data corresponding to each figure contains fold changes value in square brackets for some Phosphosites. It would be straightforward for the readers to understand if the data is represented as graphs, which would be just a fold representation of the phosphoproteins varying. The authors have Graphpad PRISM software which would this job further easy.

Response: At the suggestion of the Reviewer, we have added new diagrams in the supplementary data which show a fold representation of the phosphoproteins varying.

 

In addition, we suggest the authors to highlight the phosphoproteins which show significant variations in each pathway either positive or negative way.

Response: We agree in principle with the reviewer´s suggestion to highlight phosphoproteins that show significant differences in each pathway. However, for many proteins, it would be extremely difficult to determine only one mode of action (positive or negative) that varies in particular experimental groups. Nevertheless, we have highlighted the major phosphoproteins with significant differences in the Discussion section.

 

It would have better if the supplementary table was available as excel sheet.

Response: We fully agree with this reviewer´s comment. Unfortunately, during the submission process we encountered a technical problem in adding the supplementary table as an excel file (it was only possible to add a single file of supplementary material in pdf format). We have attempted to resolve this problem during the submission of the revised manuscript and hope that the supplementary table will be available as excel sheet.

Reviewer 3 Report

In this work Drastichova and collaborators conducted a proteomic analysis of the phosphorylation signaling elicited by the thyrotropic releasing hormone (TRH) in a cell line derived from a rat tumor of pitutitary gland (GH1). TRH effect was evaluated in the set of beta-arrestin contribution and THR-receptor triggering with two different agonists, the hormone itself and a taltirelin (TAL), which is a superagonist.  Deciphering of the phosphosignaling dynamics promoted by TRH is crucial to delineate specific pharmacological approaches and in particular in the context of biased agonism. This phenomenon describes the phenomenon that a ligand preferentially activates one of several signaling pathways, whereas another agonist in the same system and acting on the same receptor preferentially activates another pathway.  

 

The work is interesting and intended to get valuable information, nonetheless there are several issues that must be addressed to improve the manuscript.

 

Comments:

1) The first point is the manuscript is too long, the introduction and the discussion should be more concise. The role of b-arrestin as a negative regulator but also as a possible positive regulator should be described clearer in the introduction.

2) The aim and the strategy of the study should be described at the beginning of the results section, at first is difficult to understand which is the experimental design and strategy the authors follow.

3) Regarding the experimental design I see a concern here. The authors mention that in the stimulation conditions used they do not see any phosphorylation at MAPK in the discussion. I was looking for that along the results section, wondering what happen with this protein and other common residues identified in the most familiar proteins. They stated that the experimental conditions used did not trigger such activation, although TRH is reported to trigger MAPK phosphorylation. The same is also occurring with EGFR. A previous study using different concentrations of the two agonists must had been performed before, evaluating the effects by more easy technologies like western blot to select the most adequate concentration of the agonists.

Then it is very difficult to know if under those less conditions, modifications in phosphosites located in functional domains could had been found. The mention that all the alterations in the phosphosites found in the study are only in the disorderer regions must be mention briefly in the result section.

4) Another experimental limitation that the authors should mention is the use of a cancer derived cell line, which could have modifications in different signaling pathways.

5) Since the phosphor-residues identified correspond to the rat proteins, my doubt is if all the authors have included in report the corresponding equivalent residue in human.

6) Figures:

GPCR replaced by TRH-R?

The division of the data in the different signaling figures is an excellent idea. Whereas any given figure shows all the variations found, this is at some extent overwhelming. To facilitate the conclusion of each figure, perhaps a part b and c can be added to highlight in a more basic cartoon the molecules whose phoshophorylation is modified by b) beta-arrestin and c) differentially modified by TRH and TAL.

7) The authors mention that PI3K phosphorylate PAK4 (l 257). This must be corrected since PI3K is a lipid kinase.

Author Response

Response to the Reviewers´ comments

We would like to thank the reviewers for their insightful and very thorough comments on the manuscript. We have revised the manuscript accordingly. We have modified the manuscript according to the reviewers´ suggestions as much as possible. Below is our response to the issues raised in the review.

 

Reviewer 3

In this work Drastichova and collaborators conducted a proteomic analysis of the phosphorylation signaling elicited by the thyrotropic releasing hormone (TRH) in a cell line derived from a rat tumor of pituitary gland (GH1). TRH effect was evaluated in the set of beta-arrestin contribution and THR-receptor triggering with two different agonists, the hormone itself and a taltirelin (TAL), which is a superagonist. Deciphering of the phosphosignaling dynamics promoted by TRH is crucial to delineate specific pharmacological approaches and in particular in the context of biased agonism. This phenomenon describes the phenomenon that a ligand preferentially activates one of several signaling pathways, whereas another agonist in the same system and acting on the same receptor preferentially activates another pathway.  

The work is interesting and intended to get valuable information, nonetheless there are several issues that must be addressed to improve the manuscript.

 

Comments:

1) The first point is the manuscript is too long, the introduction and the discussion should be more concise. The role of b-arrestin as a negative regulator but also as a possible positive regulator should be described clearer in the introduction.

Response: We agree with the Reviewer´s comment that the manuscript is unusually long. However, this study is very complex because it is based on a large data set that we would prefer not to reduce because it would be very difficult to determine appropriate criteria for further selection of proteins involved in selected biological processes. If we were to select only proteins with phosphorylation sites with known functions, we would have no possibility of finding new protein phosphorylation sites involved in TRH-R-mediated signaling. The results of our present study encompass several topics that may be of interest to a broader group of researchers. Therefore, we believe that the length of the Introduction and Discussion sections is appropriate. One of reasons we chose the journal Cells to submit this manuscript is that this journal (as stated in the journal aims) encourages researchers to publish their results in as much detail as possible without limiting the length of articles and accepts manuscripts that communicate research with impact to a broader audience. In accordance with the Reviewer´s recommendation, we have mentioned the role of β-arrestin2 in positive and negative regulation in the Introduction section of the revised manuscript.

 

2) The aim and the strategy of the study should be described at the beginning of the results section, at first is difficult to understand which is the experimental design and strategy the authors follow.

Response: According to the Reviewer´s recommendation, we have described the aim and the strategy of the study at the beginning of the Results section of the revised manuscript.

 

3) Regarding the experimental design I see a concern here. The authors mention that in the stimulation conditions used they do not see any phosphorylation at MAPK in the discussion. I was looking for that along the results section, wondering what happen with this protein and other common residues identified in the most familiar proteins. They stated that the experimental conditions used did not trigger such activation, although TRH is reported to trigger MAPK phosphorylation. The same is also occurring with EGFR. A previous study using different concentrations of the two agonists must had been performed before, evaluating the effects by more easy technologies like western blot to select the most adequate concentration of the agonists.

Then it is very difficult to know if under those less conditions, modifications in phosphosites located in functional domains could had been found. The mention that all the alterations in the phosphosites found in the study are only in the disorderer regions must be mention briefly in the result section.

Response: We thank the Reviewer for these comments. Actually, our aim was not to confirm previous findings that activation of TRH-R triggers phosphorylation and activation of ERK and EGFR. This would not provide any new insights. We are convinced that not only the ligand alone but also its concentration can determine which signaling pathways are triggered and in what way they are affected. We did not focus on specific signaling pathways, but we were interested in looking for changes in postreceptor signaling pathways that have not been previously described in the context of TRH-R activation. It should be mentioned that not all phosphorylations were found in disordered regions. The phosphorylations found in the functional and binding domains are described in the Results section. The location of many phosphorylations in disordered regions suggests that signal transduction in many cases occurs via phosphorylation of phosphosites in disordered regions, the precise role of which is not yet known. In agreement with the Reviewer recommendation, we briefly mentioned in the revised manuscript that only some phosphorylations occurred in functional and binding domains and that many phosphorylations were located in disordered regions.

 

4) Another experimental limitation that the authors should mention is the use of a cancer derived cell line, which could have modifications in different signaling pathways.

Response: In accordance with the Reviewer recommendation, we have included information about this limitation in the Discussion section of the manuscript.

 

5) Since the phosphor-residues identified correspond to the rat proteins, my doubt is if all the authors have included in report the corresponding equivalent residue in human.

Response: We fully understand the Reviewer´s concerns, but our goal was not to compare all phosphor-residues in the rat with the corresponding equivalent residues in human. We focused only on the phosphor-residues whose human equivalents are known to be localized in some functional domains or to have certain specific functions. For those interested in a detailed comparison of the phosphor-residues, it is easily possible to compare the rat sequence with the human sequence of a specific phosphoprotein using the sequence tool (SIM Alignment Tool - Protein Sequences (expasy.org)). For this purpose, we have provided the protein ID from the UniProt database for each protein in the corresponding figures. Performing systematic comparisons for all phosphor-residues mentioned in this study would require at least two more weeks of intensive work. However, we may consider performing such comparisons in future studies.

 

6) Figures: GPCR replaced by TRH-R?

The division of the data in the different signaling figures is an excellent idea. Whereas any given figure shows all the variations found, this is at some extent overwhelming. To facilitate the conclusion of each figure, perhaps a part b and c can be added to highlight in a more basic cartoon the molecules whose phoshophorylation is modified by b) beta-arrestin and c) differentially modified by TRH and TAL.

Response: We appreciate these suggestions by the Reviewer. We have replaced the term GPCR with TRH-R in the figures of the revised manuscript. We think it is a good idea to divide the experimental groups into additional figures, but we are concerned that this will result in the loss of information that is now easy to read, in which the experimental groups show individual phosphoproteins that are differentially phosphorylated. Moreover, the creation of such figures is very time-consuming. It would hardly be feasible to divide eleven figures into parts within the 10 days allotted for the revision. Apart from that, such a change would increase the length of the manuscript enormously (and the length of the manuscript has already been criticised). Nevertheless, we will consider using such a scheme of figure preparation in future work with smaller data sets of phosphoproteins.

 

7) The authors mention that PI3K phosphorylate PAK4 (l 257). This must be corrected since PI3K is a lipid kinase.

Response: We thank the Reviewer for pointing out this error. The incorrect statement regarding PAK4 phosphorylation has been replaced by the more appropriate statement that PI3K signaling promotes phosphorylation of PAK4 at Ser181 (Annunziata et al. Int. J. Mol. Sci. 2020, 21, 3818; doi:10.3390/ijms21113818). This statement was also corrected in Figure 1 of the revised manuscript.

Round 2

Reviewer 1 Report

The revised manuscript has been improved to some extent as was suggested by Reviewer's. There is still impression that the manuscript is too long and contain too many References. Nevertheless there should be added some information before paper publication:

1. It is not stated in Materials and methods how WB results were calculated (how many independent experiments, which software was used etc.)  The silencing efficiency should be shown also in histograms (Fig.S1).
2. In Material and method section (chapter 2.2) lack of justificatio of chosen concetrations of TRH and TAL (1 microM seems be high, not physiologically relevant). Moreover it is not clear under such experimental conditions (medium content) the experiments were performed. There is a doubt that if FBS or HS were present in experimental medium then metabolic stability of TRH or TAL could be somehow affected by this factor to the different extent. This issue should be clarified.

Author Response

Response to the Reviewers´ comments

We would like to thank the reviewers for their critical comments on the manuscript. We have revised the manuscript accordingly.

 

 

Reviewer 1

The revised manuscript has been improved to some extent as was suggested by Reviewer's. There is still impression that the manuscript is too long and contain too many References. Nevertheless there should be added some information before paper publication:

1. It is not stated in Materials and methods how WB results were calculated (how many independent experiments, which software was used etc.). The silencing efficiency should be shown also in histograms (Fig.S1).

Response: According to the reviewer´s suggestion, we have added new paragraph dealing with Western blotting and evaluation of results in Materials and Methods of the revised manuscript. We have also added new histograms to Figure S1 showing the silencing efficiency.

 

2. In Material and method section (chapter 2.2) lack of justification of chosen concentrations of TRH and TAL (1 microM seems be high, not physiologically relevant). Moreover it is not clear under such experimental conditions (medium content) the experiments were performed. There is a doubt that if FBS or HS were present in experimental medium then metabolic stability of TRH or TAL could be somehow affected by this factor to the different extent. This issue should be clarified.

Response: In accordance with the reviewer´s suggestion, we have specified in more detail the experimental conditions used for treatment of cells with TRH or TAL. We agree with the reviewer´s comment that FBS or HS could potentially affect metabolic stability of TRH or TAL. Indeed, growth medium was replaced with medium supplemented with 1% FBS before the start of the experiment. This information was included in the Material and Methods section of the revised manuscript. Cells were treated with 1 mM TRH or TAL to ensure that a maximal response to stimulation was elicited. TRH and its analogs have been used at a concentration range of 0.1-100 mM in many previous studies (e.g., Narumi et al., 1983, Life Sci. 32:1637-1645; Fehrer et al., 1985, Gen. Comp. Endocrinol. 59:64-72; López-Barneo et al., 1990, Proc. NatI. Acad. Sci. USA 87:8150-8154; Ishigara et al., 1991, Brain Res. 554:203-208; Benítez et al., 1995, Neurosci. Lett. 187(1):37-40; Wilber and Xu, 1998, Thyroid 8:897-901; Drmota et al., 1998, J. Biol. Chem. 273:21699-21707: Galas et al., 1999, Endocrinology 140:3264-3272; Pesanova et al., 1999, FEBS Lett. 464:35-40; Kubera et al., 2000, Pol. J. Pharmacol. 52:481-486; Pu and Liu, 2002, J. Cell. Biochem. 86:268-276; Urayama et al., 2003, J. Pharm. Pharmacol. 55:603-608.; Luo and Stopa, 2004, J. Alzheimer’s Dis. 6:527–536; Drastichova et al., 2010, J. Cell. Biochem. 109:255-264). 

Reviewer 2 Report

The authors have addressed all the issues raised by the reviewer. The discussion has been improved significantly improved. Authors improved the supplementary data which helps readers of specific interest.

Author Response

We appreciate very much the reviewers positivive evaluation of the revised manusript.

Reviewer 3 Report

The authors modified the manuscript to address some of the points I  suggested. In general, the manuscript has improved, but it is still too long, and  the information provided is very difficult to follow. It is also difficult to drag quick conclusions based in the variation of the phosphoresidues among species.

The article requires some editing, mainly to check that all the phrases have sense in the context of the text. Please double check that in all the cases if the phosphoresidues identified in the functional or partner binding regions are mentioned. The human homologs should be mentioned for these cases. 

Specific comments (the numbers correspond to the previous list of commentaries).

1) OK, although the manuscript is still too long.

2) The authors have mentioned the aim and the strategy at the beginning of the results section. However the statements are still confusing. The characteristics of the cell line used should be mentioned here to understand the general strategy. L-220

3 and 5) Localization of the modified phosphoresidues in the protein. The authors mention now that only some of the phosphoresidues identified are localized in functional or binding domains and that they are directly mentioned (L266). I think their equivalent in human should be mentioned too in order to make this information easily extrapolated, example L314, L341, 342 and else.

4) OK

6) OK

7) The authors corrected the mistake but still the phrase has no sense, please corrected. L 339.

Please double check to verified mistakes like that in the manuscript.

Author Response

Response to the Reviewers´ comments

We would like to thank the reviewers for their critical comments on the manuscript. We have revised the manuscript accordingly.

Reviewer 3

The authors modified the manuscript to address some of the points I suggested. In general, the manuscript has improved, but it is still too long, and  the information provided is very difficult to follow. It is also difficult to drag quick conclusions based in the variation of the phosphoresidues among species.

The article requires some editing, mainly to check that all the phrases have sense in the context of the text. Please double check that in all the cases if the phosphoresidues identified in the functional or partner binding regions are mentioned. The human homologs should be mentioned for these cases. 

Response: In accordance with the reviewer´s recommendation, we have reviewed all the wording in the revised manuscript and we are convinced that it is reasonable. The human homologs were included in the revised manuscript.

Specific comments (the numbers correspond to the previous list of commentaries).

1) OK, although the manuscript is still too long.

Response: We agree with the reviewer´s comment that the manuscript is relatively long. However, it should be kept in mind that it is a complex study based on a huge amount of data, and therefore it is difficult to describe and discuss the results only briefly. For this reason we chose the journal Cells to submit this manuscript because it encourages researchers to publish their results in as much detail as possible without limiting the length of articles.

2) The authors have mentioned the aim and the strategy at the beginning of the results section. However the statements are still confusing. The characteristics of the cell line used should be mentioned here to understand the general strategy. L-220

3 and 5) Localization of the modified phosphoresidues in the protein. The authors mention now that only some of the phosphoresidues identified are localized in functional or binding domains and that they are directly mentioned (L266). I think their equivalent in human should be mentioned too in order to make this information easily extrapolated, example L314, L341, 342 and else.

Response: In accordance with the reviewer´s suggestions, we have changed the description of the aim and the strategy of the study at the beginning of the Results section of the revised manuscript. We have added human equivalents to the rat phosphoresidues localized in the functional or binding domains. We verified that all phosphoresidues identified in the functional or partner binding regions are correctly indicated in the revised manuscript.

4) OK

6) OK

7) The authors corrected the mistake but still the phrase has no sense, please corrected. L 339.

Response: In accordance with the Reviewer’s recommendation, we have changed some of this wording. But we really do not understand why the original phrase did not make sense. We believe that this reviewer´s comment is probably based on a misunderstanding. The reviewer stated in the first round of revisions that PI3K is a lipid kinase and that the phrase is incorrect. We made a conscious effort to comply with the reviewer’s request. It should be pointed out that PI3K is lipid/protein kinase with dual specificity. The p110 subunit of PI3K has protein-serine kinase activity with specificity for the PI3K subunit p85 (Dhand R. et al., 1994, EMBO J. 13(3), 552-533). PI3K also phosphorylates Ser585 in interleukin-3 and granulocyte-macrophage colony-stimulating factor receptors (Thomas D. et al., 2013, PLOS Biol., 11(3), e1001515), indicating that PI3K can phosphorylate various proteins at their serine residues. PAK4 has been shown to interact directly with PI3K subunit p85 (PIK3R) and both are regulators of PI3K/Akt pathway (King H. et al. 2017, Sci. Rep. 7, 42575; Haapalainen, A.M. et al., 2021, J Cell. Mol. Med. 25(13), 6304-6317). There is evidence that PI3K signaling promotes PAK4 activation and phosphorylation at Ser181 in the 14-3-3 binding motif (Annunziata M. et al., Int. J. Mol. Sci. 2020, 21, 3818) and it cannot be even ruled out that Ser181 in PAK4 is directly phosphorylated by PI3K. We hope that this issue is now clearer.