Exploring Protein Kinase CK2 Substrate Recognition and the Dynamic Response of Substrate Phosphorylation to Kinase Modulation
Round 1
Reviewer 1 Report
This manuscript by Cesaro and colleagues discusses the growing landscape of cellular CK2 substrates and outlines complementary strategies for identifying bonafide physiological CK2 substrates. The manuscript makes a number of important points regarding the different strategies for manipulating CK2 activity and the limitations of these strategies. Accordingly, the manuscript has the potential to be an important contribution to the field. However, in its present form, there is considerable room for improvement as there are many grammatical and/or typographical errors that are confusing for the reader. As a general comment, the manuscript would benefit significantly from careful proofreading and editing – especially for the latter half of the manuscript.
Additional points.
Line 69 - proline-directed instead of prolino-directed
Line 126 – The term “more reliable” within the statement regarding the utility of the CK2 consensus for identifying direct substrates for CK2 seems too strong from my perspective. While there is no question that the consensus has led to the identification of many novel CK2 substrates, I would recommend removing “and more reliable” from the statement.
Line 133 – differ rather than differs?
Line 143 – Similar to my comment regarding line 126, I would suggest replacing “The more reliable” with a phrase such as “One potential approach…”
Line 163. I appreciated the statement regarding the potential for misleading results arising from transient over-expression. This is an important point.
Line 201. I did not fully understand the statement “The rate of CK2 inhibition…”. I am not sure if “rate of inhibition” is what is meant. Furthermore, it is not entirely clear what will be learned by performing kinase assays with cell lysates when using non-covalent inhibitors that may not bind to the kinase to same extent in an extract as is the case within intact cells. Overall, I believe this section needs to be clarified.
Line 217 – should “crescent dose” be “increasing dose”?
Beyond line 217, I have not highlighted specific issues related to the quality of the writing since there was an increase in the number of grammatical and/or typographical issues that need to be addressed.
As noted above, there is considerable room for improvement as there are many grammatical and/or typographical errors that are confusing for the reader. As a general comment, the manuscript would benefit significantly from careful proofreading and editing – especially for the latter half of the manuscript.
Author Response
REVIEWER 1
This manuscript by Cesaro and colleagues discusses the growing landscape of cellular CK2 substrates and outlines complementary strategies for identifying bonafide physiological CK2 substrates. The manuscript makes a number of important points regarding the different strategies for manipulating CK2 activity and the limitations of these strategies. Accordingly, the manuscript has the potential to be an important contribution to the field. However, in its present form, there is considerable room for improvement as there are many grammatical and/or typographical errors that are confusing for the reader. As a general comment, the manuscript would benefit significantly from careful proofreading and editing – especially for the latter half of the manuscript.
The manuscript has been proofread by an English expert.
Additional points.
Line 69 - proline-directed instead of prolino-directed
Done.
Line 126 – The term “more reliable” within the statement regarding the utility of the CK2 consensus for identifying direct substrates for CK2 seems too strong from my perspective. While there is no question that the consensus has led to the identification of many novel CK2 substrates, I would recommend removing “and more reliable” from the statement.
Done.
Line 133 – differ rather than differs?
Done.
Line 143 – Similar to my comment regarding line 126, I would suggest replacing “The more reliable” with a phrase such as “One potential approach…”
Done.
Line 163. I appreciated the statement regarding the potential for misleading results arising from transient over-expression. This is an important point.
Line 201. I did not fully understand the statement “The rate of CK2 inhibition…”. I am not sure if “rate of inhibition” is what is meant.
“The rate of inhibition” has been changed in “the degree of inhibition”.
Furthermore, it is not entirely clear what will be learned by performing kinase assays with cell lysates when using non-covalent inhibitors that may not bind to the kinase to same extent in an extract as is the case within intact cells. Overall, I believe this section needs to be clarified.
We have added a new sentence and reference describing that this assay works also for non-covalent inhibitors
Line 217 – should “crescent dose” be “increasing dose”?
Done.
Beyond line 217, I have not highlighted specific issues related to the quality of the writing since there was an increase in the number of grammatical and/or typographical issues that need to be addressed.
Comments on the Quality of English Language
As noted above, there is considerable room for improvement as there are many grammatical and/or typographical errors that are confusing for the reader. As a general comment, the manuscript would benefit significantly from careful proofreading and editing – especially for the latter half of the manuscript.
The manuscript has been proofread by an English expert.
Reviewer 2 Report
The manuscript is submitted as a review by the group of Mauro Salvi on „Exploring Protein Kinase CK2 Substrate Recognition and the Dynamic Response of Substrate Phosphorylation to Kinase Modulation“. The topic as such is of high interest and the group presenting the manuscript has published over the years many original studies and reviews on CK2 with high impact and considerable response in the community. From this regard, the group appears predestined to provide a review on the issue as given, however with the manuscript as presented, I see some problems.
The paper is supposed to be a review, but contains a considerable part of original research. As given in Fig. 1, the authors have analysed the CK2 consensus (substrate recognition) sequence applying more recently available software tools and come to a new (?) logo of the CK2 consensus sequence as to be S/T[E/D]x[E/D]. They write “… the substrate logo (they) generated in 2009 with a lower number of substrates (433) exhibits significant similarity to the logo generated in this study“. But what does this mean? Is it identical or not, and in case that it is not, does it lead to a different pattern of putative substrates?
Similar as the results presented in Fig1, the results as given in Fig. 2 (phosphorylation of selected substrates in dependency of a – selective – CK2 inhibitor and Fig. 3 (phosphorylation of substrates in dependency of down regulation or knock out of distinct CK2 subunits) appear to be highly interesting. But if I understood correctly, they are not cited from other original research paper, but integrated as original research within the context of a review.
In case of a review, the manuscript appears somehow incomplete at some points, e.g. in chapter 3 “the specific tools” for the “assessment of site-specific phosphorylation in a protein“ are said to be given. But Mass Spec approaches as described e.g. in Caefer et al., (Front Mol Biosci 2022, 9:850661) or phosphoproteomics as described in reference [49], are completely missing.
Minor point: the manuscript could benefit from severe proof reading. Some sentences are incomplete, some mix up singular and plural etc., but this is not a problem in general.
My suggestion would be to decide firstly whether to submit a review or a research paper (my preference goes clearly towards research paper), and reorganize the manuscript accordingly.
In case of a review, it would be recommendable to emphasize what is new to this review and has not been described in other reviews on CK2 before.
The manuscript would significantly benefit from thorough proof reading, eg. by the proof reading service provided by MDPI.
Author Response
REVIEWER 2
The manuscript is submitted as a review by the group of Mauro Salvi on „Exploring Protein Kinase CK2 Substrate Recognition and the Dynamic Response of Substrate Phosphorylation to Kinase Modulation“. The topic as such is of high interest and the group presenting the manuscript has published over the years many original studies and reviews on CK2 with high impact and considerable response in the community. From this regard, the group appears predestined to provide a review on the issue as given, however with the manuscript as presented, I see some problems.
The paper is supposed to be a review, but contains a considerable part of original research. As given in Fig. 1, the authors have analysed the CK2 consensus (substrate recognition) sequence applying more recently available software tools and come to a new (?) logo of the CK2 consensus sequence as to be S/T[E/D]x[E/D]. They write “… the substrate logo (they) generated in 2009 with a lower number of substrates (433) exhibits significant similarity to the logo generated in this study“. But what does this mean? Is it identical or not, and in case that it is not, does it lead to a different pattern of putative substrates?
The manuscript has been proofread by an English expert.
Similar as the results presented in Fig1, the results as given in Fig. 2 (phosphorylation of selected substrates in dependency of a – selective – CK2 inhibitor and Fig. 3 (phosphorylation of substrates in dependency of down regulation or knock out of distinct CK2 subunits) appear to be highly interesting. But if I understood correctly, they are not cited from other original research paper, but integrated as original research within the context of a review.
We have now added the original research references.
In case of a review, the manuscript appears somehow incomplete at some points, e.g. in chapter 3 “the specific tools” for the “assessment of site-specific phosphorylation in a protein“ are said to be given. But Mass Spec approaches as described e.g. in Caefer et al., (Front Mol Biosci 2022, 9:850661) or phosphoproteomics as described in reference [49], are completely missing.
Regarding Chapter 3 we have now added a sentence regarding the mass spectrometry approach to phosphosites detection as suggested. However, as stated in the review a full discussion of these techniques and their applications is beyond the scope of this review.
My suggestion would be to decide firstly whether to submit a review or a research paper (my preference goes clearly towards research paper), and reorganize the manuscript accordingly.
In case of a review, it would be recommendable to emphasize what is new to this review and has not been described in other reviews on CK2 before.
We thank the reviewer for the suggestion, but we prefer a review format because in the presented experiments we highlight some important observations that have been made, at least partially, in other publications. We decide to repeat some experiments here under more complete conditions in order to better clarify and discuss these observations. We have followed the reviewer's suggestion and added the original references.
Minor point: the manuscript could benefit from severe proof reading. Some sentences are incomplete, some mix up singular and plural etc., but this is not a problem in general.
The manuscript has been proofread by an English expert.
Reviewer 3 Report
In this article, the authors reviewed about the recent evidence of how protein kinase CK2 (CK2) recognizes its substrates. In addition, the authors discussed about the pros and cons of the existing methodologies used to manipulate CK2 activity in cells and explored the dynamic response of substrate phosphorylation to CK2 modulation.
# Comments:
1) First, the authors should accurately define “CK2”; does “CK2” means “casein kinase II”?
2) The scale bars are necessary for all immunoblotting data.
3) In figure 3A, it would be better to move the bands of calnexin into lowest position.
4) The authors should summarize and indicates current pharmacological CK2 inhibitors into the figures and tables indicating their structures, molecular features (especially the effects upon each subunit of CK2) and referenced papers.
5) If the authors review about knockdown of CK2, the authors should also get in touch with microRNAs and long noncoding RNAs which regulate CK2 expression and CK2 related molecular signaling; indicating this information by the figures and tables is also mandatory.
Author Response
REVIEWER 3
In this article, the authors reviewed about the recent evidence of how protein kinase CK2 (CK2) recognizes its substrates. In addition, the authors discussed about the pros and cons of the existing methodologies used to manipulate CK2 activity in cells and explored the dynamic response of substrate phosphorylation to CK2 modulation.
# Comments:
1) First, the authors should accurately define “CK2”; does “CK2” means “casein kinase II”?
Done in the abstract.
2) The scale bars are necessary for all immunoblotting data.
Done.
3) In figure 3A, it would be better to move the bands of calnexin into lowest position.
Done.
4) The authors should summarize and indicates current pharmacological CK2 inhibitors into the figures and tables indicating their structures, molecular features (especially the effects upon each subunit of CK2) and referenced papers.
A new table describing the most commonly used CK2 pharmacological inhibitors has been added as requested.
5) If the authors review about knockdown of CK2, the authors should also get in touch with microRNAs and long noncoding RNAs which regulate CK2 expression and CK2 related molecular signaling; indicating this information by the figures and tables is also mandatory.
We have introduced a new sentence describing the different RNA interference platforms.
Reviewer 4 Report
please see the attached file
Comments for author File: 
Comments.pdf
please see the attached file (Minor comments)
Author Response
REVIEWER 4
Major comments:
The authors presented an interesting, concise and comprehensive review summarizing the landscape of CK2 substrates/phosphosites and dynamics of the casein kinase 2 modulation. The review provides the most important information about CK2 sequence/structure, substrate recognition and phosphorylation, mentions the methods for studying the protein kinase activity/substrate specificity and critically discusses the available approaches to target the expression/activity of this kinase. The following comments are raised by the Reviewer that the Authors should address when revising their manuscript.
Figure 1A. Please provide the Protein Data Bank ID (code) for the presented CK structure and what peptide substrates are bound (what are green and what red colors?). To be more specific, the Figure 1A could be: “Electrostatic potential mapped onto the molecular surface of CK2 (PDB ID: ??????) and the binding of a peptide substrate (what peptide and what red and green mean?). Red: negative; blue: positive. Or provide the color scale bar. And what software was used to calculate the electrostatic potential? Please correct accordingly.
Done
The Authors should add a table summarizing the available inhibitors along with the information on their structure, binding region within the CK2, mode of action (ATP-competitive, allosteric?), IC50 etc. This would be a great added value of this manuscript.
Done
How many CK2 mutants have been characterized so far and how their use may help in exploring the phosphosite landscape of CK2 and dynamic regulation of the protein kinase activity? The Authors are encouraged to discuss this interesting aspect in more detail as they only mentioned it in Conclusions.
Done as requested.
Minor comments:
1. Lane 10: Please replace “ser/thr” with “serine/threonine” (as first time mentioned), and also define CK2 as “casein kinase II (or 2)” in the Abstract.
Done.
Key words: please add “casein kinase 2”.
Done.
Lane 26: What Authors mean by “phosphosites substrates”. Do they refer to different proteins that may be phosphorylated by CK2 (substrates) or different phosphorylation sites (phosphosites) that may be one or more on the same protein substrate. Please be more specific. “phosphosites” alone (or “substrates” alone) would be more accurate .
Done.
Lane 39: “the cell secretome” in this context is not precise. “secreted phosphoproteins” would bemore accurate, as secretome is a specific set of all secreted proteins under specific conditions(as a cell feature).
Done.
Lane 42: please write the gene names in italics (https://www.gmb.org.br/geneprotein-
nomenclature-guidelines).
Done.
Lane 101: please replace “it” with “its”.
Done.
Lane 101: please correct the sentence, e.g. for “The Weblogo analysis of CK2 phosphosites presented in Fig. 1B is the...”
Done.
Lane 111: please correct the sentence, e.g. “In our work from 2009 [17],...”
Done.
Lane 113-114: Please improve the language of the sentence “We also...” It is not
incomprehensible.
Done.
Lane 121: Please change “Cristal” with “Crystal”.
Done.
Lane 128: Please correct the English and scientific soundness of the chapter 3 title. What Authors meant by “matching”? Please correct.
Done.
Lane 133, 137: please replace “differs” with differ”, and “refers” with “refer”.
Done
Lane 139-142: please improve the language. “phosphorylation of a specific site in cell”?. Be more specific.
Done
Lane 149: please replace “whose”, with “, of which”.
Done
Lane 155: please replace “critical” with “critically”.
Done
Lane 159: changing “substrate” for e.g. “target proteins” might be more accurate in the context of the functional implications of the CK2 activity modulation discussed here.
Done
Lane 165: please replace “kinase” with “kinases”.
Done
Lane 168: please correct as follows “knockdown/knockout of the protein kinase expression”. Or protein-encoding gene.
Done
Lane 174-177: please improve the language, e.g. “The inhibitors most used in the past, including 4,5,6,7-tetrabromobenzotriazole (TBB) or 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT), revealed a lot of off-target effects and have been largely dismissed (for a recent review on CK2 inhibitors see [39]).”
Done
Lane 1177-179: please improve the language. “respect to” (perhaps “with regard to”?), and what Authors mean by “the inhibitor of reference of targeting CK2”?
Done
Lane 189: please replace “the use at least” with “the use of at least”.
Done
Lane 190: please replace “support” with “supports”.
Done
Lane 200: please replace “inhibition” with “inhibition efficiency”.
Done
Lane 194: “depending on the type of substrate” instead of “according...” would be a better choice.
Done
Lane 228-229: these methods target the gene encoding the protein, so to be specific the Authors should write: gene knockdown and gene knockout, and as the result of these genetic manipulations, there is a reduction or a complete loss of protein expression. Please correct.
Done
Lane 230: RNA interference (RNAi) for gene silencing can be achieved with diverse RNAi platforms, including small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). And the most common (because the easiest, vector-free mode) is siRNA. No studies using shRNA-plasmids were reported for targeting CK2? Please correct accordingly.
Done
Lane 238: please provide the references.
As per the journal's policy, the sentence has been removed due to it being based on personal communication.
Lane 242: please replace “dose” with “doses of”; Lane 250: “SiRNA” with “siRNA”.
Done
Lane 244, 306: please replace “lysates” with “lysed”. Or cell lysates were prepared as
described...
Done
Lane 248: please replace with “,and Calnexin (sc-46669) and GAPDH (ABS16) as the loading controls”.
Done
Lane 263: please replace ”We first generated” with “We generated for the first time”.
Done
Lane 264: please replace “type” with “types”.
Done
Lane 271: please replace “abolish” with “abolition” or “deletion”.
Done
Lane 277: please replace “whose” with “which”.
Done
Lane 283: please replace “an” with “a”.
Done
Lane 287: please replace “changes” with “change”.
Done
Lane 288: please replace “Fort example” with “For example,”
Done
Lane289: please add comma after “contrary”.
Done
Lane 305: please replace “[57].” with “[57]).”
Done
Lane 306: please add comma after “incubation”. And please improve the language for Lane 306.
Done
Figure 3B: please correct “substrati” for “substrates”
Done
Round 2
Reviewer 1 Report
In this revised manuscript, the authors have satisfactorily addressed the issues raised in my previous review. Most notably, the authors have provided clarifications related to issues identified in the original manuscript and the quality of the presentation has been improved. At this stage, there is only additional correction that I would recommend which I believe can be performed without need for further review. From this perspective, the legend for Figure 2 refers to Panel C but not Panel B when describing immunoblots detecting phosphorylation of CK2 substrates. Furthermore, the legend contains references to "crescent doses" of SGC-CK2-1 which I believe should be "increasing doses". Once these changes are made, I believe the manuscript will be suitable for publication.
Author Response
From this perspective, the legend for Figure 2 refers to Panel C but not Panel B when describing immunoblots detecting phosphorylation of CK2 substrates.
We have now added the description of panel B.
Furthermore, the legend contains references to "crescent doses" of SGC-CK2-1 which I believe should be "increasing doses".
Done.
Reviewer 2 Report
After reading the revised version of the paper, I can state, that it is a substantial improvement concerning English language and the clarity of results shown in Fig. 1.
However, it still remains obscure to me, what part of the results in shown in Fig. 2 and Fig. 3 are original research and which are citations of other original research paper, despite the author claim to have added the original citations. In addition, they do not clarify, as requested, what is new for this review, and what has been subject of their numerous reviews before.
So, in summary, the authors prefer to present a review, containing original research of unknown origin and do not want to explain what the novelty of this review is. (Besides, a new table was included, without comment, which looks a bit strange to me. Doubting whether it is complete, at the least the values should be left-aligned instead of central.
Author Response
However, it still remains obscure to me, what part of the results in shown in Fig. 2 and Fig. 3 are original research and which are citations of other original research paper, despite the author claim to have added the original citations.
We assert that these findings are already documented in the existing literature.
Regarding Figure 2, we acknowledge the necessity to incorporate and discuss additional relevant evidence from prior studies. A new paragraph has been included to address this.
Figure 3 illustrates that substrate phosphorylation can exhibit isoform specificity. To downregulate CK2 isoforms, siRNA was employed. Nevertheless, these outcomes replicate the results obtained with CK2 knockout cells, as previously outlined in the article (lines 309-315).
“The advantages of the knockdown/knockout methods over inhibitor treatments are the ability to uncover the distinct biological roles of each CK2 subunit. Indeed if in some cases CK2 catalytic subunits are highly redundant, as above discussed, isoforms specific roles have also been described [87–89]. It has also been shown that knockout of a specific subunit may have a greater effect on the phosphorylation level of some CK2 substrates [81,82]. The same is observed with siRNA downregulation of individual CK2 subunits, as shown in Fig. 3.”
In addition, they do not clarify, as requested, what is new for this review, and what has been subject of their numerous reviews before.
We apologize for not responding during the initial revision. We now better understand the concern. Our review's core focuses on discussing the existing tools for modulating CK2 activity. We not only provide a comprehensive description of these methods but also critically evaluate them, highlighting their pros and cons. Importantly, we draw from our practical experience with these methods, including the generation of CK2 knockout clones, to provide a unique perspective. To the best of our knowledge, this perspective is novel, and we are not aware of a similar review.
However, if the reviewer is aware of a similar review, we are prepared to withdraw our submission immediately.
So, in summary, the authors prefer to present a review, containing original research of unknown origin and do not want to explain what the novelty of this review is.
We understand that the reviewer would have preferred an original article, but we assumed from the first round of reviewing that he would also accept a review, as he left this option open. The added experiments (materials and methods are in the captions) do not add anything new (as similar results are available in the literature) but have been included because they serve to reinforce concepts and better convey the message of this review.
Besides, a new table was included, without comment, which looks a bit strange to me.
The table was requested by other reviewers. A sentence was added to the manuscript to introduce the new table (see below)
Several inhibitors targeting CK2 have been identified over the years and Table 1 lists the most important compounds with their Ki/IC50 (for a comprehensive list of CK2 inhibitors please refer to [41,42]).
Lines 181-183
Doubting whether it is complete, at the least the values should be left-aligned instead of central.
The table is intentionally not exhaustive, as mentioned in the preceding sentence. This deliberate omission is due to the recent publication of several comprehensive reviews on CK2 inhibitors. We kindly request readers to consult these works for a comprehensive list of inhibitors. The inclusion of this table serves the purpose of providing a quick reference to the most commonly used CK2 inhibitors within the scientific community.
The values have been left-aligned as required by the Reviewer.
Reviewer 3 Report
REVIEWER 3
In this article, the authors reviewed about the recent evidence of how protein kinase CK2 (CK2) recognizes its substrates. In addition, the authors discussed about the pros and cons of the existing methodologies used to manipulate CK2 activity in cells and explored the dynamic response of substrate phosphorylation to CK2 modulation.
# Comments:
1) First, the authors should accurately define “CK2”; does “CK2” means “casein kinase II”?
Done in the abstract.
⇒ OK.
2) The scale bars are necessary for all immunoblotting data.
Done.
⇒ OK.
3) In figure 3A, it would be better to move the bands of calnexin into lowest position.
Done.
⇒ OK.
4) The authors should summarize and indicates current pharmacological CK2 inhibitors into the figures and tables indicating their structures, molecular features (especially the effects upon each subunit of CK2) and referenced papers.
A new table describing the most commonly used CK2 pharmacological inhibitors has been added as requested.
⇒ The information of reference papers for each inhibitor should be also included in a new table.
5) If the authors review about knockdown of CK2, the authors should also get in touch with microRNAs and long noncoding RNAs which regulate CK2 expression and CK2 related molecular signaling; indicating this information by the figures and tables is also mandatory.
We have introduced a new sentence describing the different RNA interference platforms.
⇒ I checked the new sentences added by the authors. However, the sentence did not include any concrete information of microRNAs and long noncoding RNAs which are dysregulated and affect CK2 expression in diseases. The authors should address this point with adding new table or figure.
Author Response
⇒ The information of reference papers for each inhibitor should be also included in a new table.
Done as requested
⇒ I checked the new sentences added by the authors. However, the sentence did not include any concrete information of microRNAs and long noncoding RNAs which are dysregulated and affect CK2 expression in diseases. The authors should address this point with adding new table or figure.
We apologize for omitting the table and discussion of miRNA and long coding in our previous submission. We initially did not include this information because we believed that miRNA and long coding, despite their critical physiological importance, were not commonly used tools to downregulate CK2 expression. However, we recognize the importance of providing this information for the benefit of our readers. We have now included a completely new paragraph to highlight the importance of miRNA in CK2 regulation in physiology and disease, along with relevant references, and to critically discuss their potential use as tools to modulate CK2 expression. However, we have decided not to include a table for two reasons: 1- We have already provided a similar table in a previous review (see DOI: 10.1080/10409238.2021.1908951). 2- This review focuses primarily on tools to manipulate CK2 activity, and although miRNA and long coding are physiologically relevant, they are less useful as tools in this context.
Round 3
Reviewer 3 Report
The authors responded to all my requests appropriately.
Author Response
The authors responded to all my requests appropriately.
Thanks
