Phosphorylation Codes in IRS-1 and IRS-2 Are Associated with the Activation/Inhibition of Insulin Canonical Signaling Pathways

Round 1

Reviewer 1 Report

This interesting review proposes a nomenclature of over 50 serine residues within IRS-1 that have been reported to undergo phosphorylation by a spectrum of kinases, thereby activating or inhibiting different signaling pathways. They also identify over 10 tyrosine residues in IRS-1 or IRS-2 in response to insulin, resulting in signal transduction and the subsequent activation of PI3K.

The authors claim to have written a comprehensive review where they meticulously elucidate the roles of phosphorylation events within the intricate signaling pathways governing IRS-1 and IRS-2, with a broader perspective that illuminates the complex interplay of signaling cascades, resulting in the activation of diverse intracellular pathways.

The review is coherent and very well (even elegantly) written, and is a good summary of the state of knowledge as it was 5-10 years ago.

However, a major weakness is that it reviews only 67 references, for such a broad topic. Of these, 85% are to literature between 1985 and 2013, and only 10 references covering the period 2014-2018 (refs. 9, 12, 13, 16, 21, 23, 25, 30, 42 and 63). No reference to articles or reviews written in the last 5 years.

I did a PubMed search for papers on “IRS-1 or IRS-2 phosphorylation 2019-2023” and got 4.507 results.

As an example, a review like: White MF and Kahn CR. Insulin action at a molecular level – 100 years of progress. Mol Metab (2021) 52:101304, is ignored. As another example, the authors state in lines 293-294: “This revelation introduces a novel mechanistic insight into the role of TNFa in eliciting insulin resistance”. This novel revelation occurred in 2008 (ref. 32). Another one, lines 297-298: “This observation …elucidating a novel inhibitory function…” dates back to 2005 (ref. 33).

A critical pathway downstream of IRS-1 and 2 is FOXO1, as explored in detail a.o. by Domenico Accili and colleagues at Columbia University, is ignored. The role of IRS-2 in beta cell function is also not addressed. Names like Rohit Kulkarni or Jens Brüning are other leads for searches.

I think it will take a substantial amount of work for the authors to make their review up to date, but it may be worth it if they are willing.

Author Response

Reviewer 1

 Comments and Suggestions for Authors

This interesting review proposes a nomenclature of over 50 serine residues within IRS-1 that have been reported to undergo phosphorylation by a spectrum of kinases, thereby activating or inhibiting different signaling pathways. They also identify over 10 tyrosine residues in IRS-1 or IRS-2 in response to insulin, resulting in signal transduction and the subsequent activation of PI3K.

The authors claim to have written a comprehensive review where they meticulously elucidate the roles of phosphorylation events within the intricate signaling pathways governing IRS-1 and IRS-2, with a broader perspective that illuminates the complex interplay of signaling cascades, resulting in the activation of diverse intracellular pathways.

The review is coherent and very well (even elegantly) written, and is a good summary of the state of knowledge as it was 5-10 years ago.

 Point 1: However, a major weakness is that it reviews only 67 references, for such a broad topic. Of these, 85% are to literature between 1985 and 2013, and only 10 references covering the period 2014-2018 (refs. 9, 12, 13, 16, 21, 23, 25, 30, 42 and 63). No reference to articles or reviews written in the last 5 years.

I did a PubMed search for papers on “IRS-1 or IRS-2 phosphorylation 2019-2023” and got 4.507 results.

 Response: We very much appreciate your suggestion. We added more recent references in the manuscript. Line 45, 129, 131, 176, 200, 216, 324, 369, 410. References (2) in table 1.

 Point 2: As an example, a review like: White MF and Kahn CR. Insulin action at a molecular level – 100 years of progress. Mol Metab (2021) 52:101304, is ignored.

 Response: Thanks for your comments. We included this reference in lines 216.

Point 3: As another example, the authors state in lines 293-294: “This revelation introduces a novel mechanistic insight into the role of TNFa in eliciting insulin resistance”. This novel revelation occurred in 2008 (ref. 32).

 Response: Thanks for your observation, we eliminate the word “novel” in line 319.

Point 4: Another one, lines 297-298: “This observation …elucidating a novel inhibitory function…” dates back to 2005 (ref. 33).

 Response: Thanks for your observation, we eliminate the word “novel” in line 325.

Point 5: A critical pathway downstream of IRS-1 and 2 is FOXO1, as explored in detail a.o. by Domenico Accili and colleagues at Columbia University, is ignored. The role of IRS-2 in beta cell function is also not addressed. Names like Rohit Kulkarni or Jens Brüning are other leads for searches. I think it will take a substantial amount of work for the authors to make their review up to date, but it may be worth it if they are willing.

Response: Thank you for pointing this out. According to your comments we added a paragraph from Domenico Accili at lines 125-129. In lines 129-131 we add a paragraph from Kulkarni.

Reviewer 2 Report

The review “Phosphorylation codes in IRS-1 and IRS-2 are associated with the activation/inhibition of insulin canonical signaling pathways” is devoted to the detail aspects of phosphorylation and signaling events of substrate proteins IRS-1 and IRS-2.

Minor comments:

1.      There are some misprints in the text. For example: lane 74 – ISR-1 instead of IRS-1.

2.      Insulin receptor substrates are signaling adapter proteins that transmitting signals from the receptors of  insulin receptor family. This family consists of three homologous receptors IR, IGF-IR, and IRR. Phosphorylation of IRS-1 is observed not only in the case of IR and IGF-IR (lanes 43-44), but also upon activation of IRR. Relevant links must be added to the text. (Deyev IE, et al. Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line. Biochimie. 2017 Jul;138:62-69. doi: 10.1016/j.biochi.2017.04.002; Deyev IE, et al. Insulin receptor-related receptor as an extracellular alkali sensor. Cell Metab. 2011 Jun 8;13(6):679-89. doi: 10.1016/j.cmet.2011.03.022)

minor editing is needed. For example, lane 56: The IRS family proteins exibits... should be ...exibit... There are some misprints in the text.

Author Response

Reviewer 2

Comments and Suggestions for Authors

The review “Phosphorylation codes in IRS-1 and IRS-2 are associated with the activation/inhibition of insulin canonical signaling pathways” is devoted to the detail aspects of phosphorylation and signaling events of substrate proteins IRS-1 and IRS-2.

Minor comments:

Point 1: There are some misprints in the text. For example: lane 74 – ISR-1 instead of IRS-1.

Response: We apologize because there was an error in the text, by mistake we wrote "ISR-1" instead of "IRS-1", the correction was made in lane 74.

Point 2: Insulin receptor substrates are signaling adapter proteins that transmitting signals from the receptors of insulin receptor family. This family consists of three homologous receptors IR, IGF-IR, and IRR. Phosphorylation of IRS-1 is observed not only in the case of IR and IGF-IR (lanes 43-44), but also upon activation of IRR. Relevant links must be added to the text. (Deyev IE, et al. Alkaline pH induces IRR-mediated phosphorylation of IRS-1 and actin cytoskeleton remodeling in a pancreatic beta cell line. Biochimie. 2017 Jul;138:62-69. doi: 10.1016/j.biochi.2017.04.002; Deyev IE, et al. Insulin receptor-related receptor as an extracellular alkali sensor. Cell Metab. 2011 Jun 8;13(6):679-89. doi: 10.1016/j.cmet.2011.03.022)

Response: As you correctly suggested we added this paragraph “and also upon activation of the insulin receptor-related receptor (IRR)” at lanes 44-45. You were right that IRR is also an activator of IRS-1 phosphorylation. Also, we added your suggested references that describe this transmembrane receptor.

Author Response File: Author Response.pdf

Reviewer 3 Report

This paper has great value in its collection of what’s known about IRS-1 phosphorylation all in one place.  There are a few issues that should be addressed in order to maximize the usefulness of the review.

Major points

The text on p. 5 seems to imply that serine phosphorylation of IRS-1 activates mTOR.  This is misleading and appears to be based on a misreading of ref 22.  Ref 22 shows an association of IRS-1 serine phosphorylation with “disrupted…activation” of the Akt/mTOR pathway.  Take a look at the data in ref 22.  Please clear this up, because if there aren’t data showing that IRS-1 serine phosphorylation activates mTOR, the review paper should not imply this.

Lines 192-194 state that ref 23 shows that S302 regulates insulin signaling, while in actuality ref 23 shows pretty convincingly that S302 is dispensable (i.e. doesn’t play a role) in modulation of insulin signaling.

Line 20 serving to increase or attenuate effects of insulin.   Please consider leaving the “increase” part out of the abstract.  I might be mistaken, but it seems like the only reference to increasing insulin action is for AMPK phosphorylating IRS1 on S789 (lines 261-264).  As discussed below, the association of S789 and insulin sensitivity is a correlation, and the paper (ref 29) does not address a causal role of S789.

Line 214:  this mentions that S phosphorylation of IRS-1 activates mTOR.  This does not appear to be the case (as mentioned above for ref 22.  If there are literature reports that S phosphorylation does indeed activate mTOR, please discuss these.

Lines 251-264: please acknowledge in this paragraph that ref 29 only showed an association of S789 with increased insulin signaling and that remains possible that IRS-1 phosphorylation and enhanced insulin signaling could be independent effects of AMPK activation.

Lines 265-273:  this paragraph starts by stating that IRS S phosphorylation can activate PI3K.  Ref 30 seems to indicate the opposite:  phosphorylation of S616 is associated with impaired insulin-stimulated IRS-1 tyrosine phosphorylation.  Please re-check ref 30 and adjust the text accordingly.

Line 202 correlation of IRS serine phosphorylation and mTOR phosphorylation; please consider noting that these could be independent events (JNK/jun phosphorylate IRS-1 but also independently activate Akt/mTOR).

Minor points

Figure 1.  Please consider adding orange and orange/black diamonds to the figure key.  Readers might be confused, because it’s not immediately obvious what the orange indicates.

Lines 128-158:  consider mentioning a few more details from ref 20, such as:  expression of an IRS1-S1101A mutant increased insulin-stimulated IRS-1 tyrosine phosphorylation in cells provided with amino acids.

Lines 211-212 please mention some of the metabolic/cellular outcomes associated with S636/639 phosphorylation if they are discussed in ref 26

Lines 238-250 please add literature citations for this paragraph

Lines 296-300: please add more details about the ref 33 findings on GSK3, IRS S332, and insulin signaling

IRS-2: please consider adding information regarding whether any of the IRS-2 phosphorylation sites are homologous to IRS-1 sites, especially if the upstream kinases or downstream effects are known for IRS-1 but not IRS-2

Table 1, p. 9, IRS-1 S1101:  is SK6 a typo (S6k?)

Line 388 IRS.1

Lines 265-267:  please check grammar (maybe “phosphorylation that occurs” would work

Author Response

Reviewer 3

Comments and Suggestions for Authors

Major points

Point 1: This paper has great value in its collection of what’s known about IRS-1 phosphorylation all in one place.  There are a few issues that should be addressed in order to maximize the usefulness of the review.

 Response: We thank the reviewer for the positive evaluation of our manuscript. We believe that the review has a very important value in the knowledge of the phosphorylations of IRS-1.

Point 2: The text on p. 5 seems to imply that serine phosphorylation of IRS-1 activates mTOR.  This is misleading and appears to be based on a misreading of ref 22.  Ref 22 shows an association of IRS-1 serine phosphorylation with “disrupted…activation” of the Akt/mTOR pathway.  Take a look at the data in ref 22.  Please clear this up, because if there aren’t data showing that IRS-1 serine phosphorylation activates mTOR, the review paper should not imply this.

Response: Thank you for your comments. We correct the misinterpretation and rely on the data presented in reference 22 giving the correct meaning. The correction was made in lines 198-211. The corrections were highlighted in the manuscript.

Point 3: Lines 192-194 state that ref 23 shows that S302 regulates insulin signaling, while in actuality ref 23 shows pretty convincingly that S302 is dispensable (i.e. doesn’t play a role) in modulation of insulin signaling.

Response: Thank you so much for your valuable comment. We agree with this comment. Therefore, we have corrected the meaning of these results according to reference 22. We make sure to clarify that S302 is dispensable in the modulation of insulin signaling. The correction was made in lines 211-214. The corrections were highlighted in the manuscript.

Point 4: Line 20 serving to increase or attenuate effects of insulin.   Please consider leaving the “increase” part out of the abstract.  I might be mistaken, but it seems like the only reference to increasing insulin action is for AMPK phosphorylating IRS1 on S789 (lines 261-264).  As discussed below, the association of S789 and insulin sensitivity is a correlation, and the paper (ref 29) does not address a causal role of S789.

Response: Thank you for pointing this out. We removed the word “increase” in line 20 because this effect has not been proven. Only IRS serine phosphorylation has been shown to attenuate the effects of the signaling pathway. We made the correction at line 277.

Point 5: Line 214:  this mentions that S phosphorylation of IRS-1 activates mTOR.  This does not appear to be the case (as mentioned above for ref 22.  If there are literature reports that S phosphorylation does indeed activate mTOR, please discuss these.

Response: Thank you for your comment. We have changed these data, clarifying that serine phosphorylation of IRS 1 does not directly activate the mTOR signaling cascade. Only, the phosphorylation on serine residues of IRS-1 could play a role in the activation of the mTOR pathway. Corrections were made at lines 238-239.

Point 6: Lines 251-264: please acknowledge in this paragraph that ref 29 only showed an association of S789 with increased insulin signaling and that remains possible that IRS-1 phosphorylation and enhanced insulin signaling could be independent effects of AMPK activation.

Response: We appreciate your valuable comments. We completely agree with you. We have clarified in the text that the phosphorylation of IRS-1 and enhanced insulin signaling may be independent effects of AMPK activation, so more research is needed on this matter. We made the corrections at lines 288-290.

 Point 7: Lines 265-273:  this paragraph starts by stating that IRS S phosphorylation can activate PI3K.  Ref 30 seems to indicate the opposite:  phosphorylation of S616 is associated with impaired insulin-stimulated IRS-1 tyrosine phosphorylation.  Please re-check ref 30 and adjust the text accordingly.

Response: Thank you for your valuable comments. We adjusted the text according to reference 39. The text was as follows: Several studies suggest that phosphorylation occurs at a specific serine residue within IRS-1 and could play a role in the activation of both the PI3K and MAPK signaling pathways. Specifically, IRS-1 Ser616 has been identified as susceptible to phosphorylation induced by 20-hydroxyeicosatetraenoic acid (20-HETE), a bioactive lipid mediator known to elicit endothelial dysfunction. This phosphorylation event is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway. We made the corrections at lines 291-297.

 Point 8: Line 202 correlation of IRS serine phosphorylation and mTOR phosphorylation; please consider noting that these could be independent events (JNK/jun phosphorylate IRS-1 but also independently activate Akt/mTOR).

Response: Thank you for your comments. We agree with you. We consider that the phosphorylation of serine residues on IRS-1 and the activation of the mTOR pathway could be independent events. But there is evidence that serine phosphorylation in IRS could play an important role and converge at some point with the mTOR pathway, however, more research is needed in this regard. Correction was made at line 225.

Minor points

Point 9: Figure 1.  Please consider adding orange and orange/black diamonds to the figure key.  Readers might be confused, because it’s not immediately obvious what the orange indicates.

 Response: Thank you for your comments. We add orange and orange/black diamonds to the figure key. We believe that with this modification, the meaning of these symbols is more easily understood.

Point 10: Lines 128-158:  consider mentioning a few more details from ref 20, such as: expression of an IRS1-S1101A mutant increased insulin-stimulated IRS-1 tyrosine phosphorylation in cells provided with amino acids.

Response: Thank you for your observations. We add more details in the text from reference 20. In their in vitro assays S6K1 phosphorylates IRS-1 at Ser1101 and the mutation of Serine to Alanine of this site largely blocks the ability of amino acids to abolish IRS-1 tyrosine and Akt phosphorylation. In CHO cells expressing the HA-IRS-1 Ser1101 incubated with insulin and amino acids showed increased phosphorylation of Ser1101 compared with the mutant HA-IRS-1 Ser1101Ala, while tyrosine phosphorylation was higher in the mutant. Lines 164-169.

Point 11: Lines 211-212 please mention some of the metabolic/cellular outcomes associated with S636/639 phosphorylation if they are discussed in ref 26 (Tzatsos)

Response: Thank you for your comments. We add more details about the metabolic/cellular outcomes associated with S636/639 phosphorylation in the text from reference 28. Nevertheless, the author does not mention the metabolic and cellular outcomes of the phosphorylation of IRS-1 Ser636/639. However, we wanted to add more details of IRS-1 Ser636/639 phosphorylation. Corrections were made at lines 234-237.

Point 12: Lines 238-250 please add literature citations for this paragraph.

Response: We appreciate your comments. We added the references in the paragraph and made a correction at lines 269-270.

Point 13: Lines 296-300: please add more details about the ref 33 findings on GSK3, IRS S332, and insulin signaling.

Response: Thank you for this suggestion. Sorry for not describing it before. We have added more details in lines 323-324 and 326-330.

Point 14: IRS-2: please consider adding information regarding whether any of the IRS-2 phosphorylation sites are homologous to IRS-1 sites, especially if the upstream kinases or downstream effects are known for IRS-1 but not IRS-2.

Response: Thank you very much for pointing this out. We have added the amino acids with homology between IRS-1 and IRS-2. Corrections were made at lines 366-369 and 407-410.

Point 15: Table 1, p. 9, IRS-1 S1101:  is SK6 a typo (S6k?)

Response: Thank you very much for pointing this out. We have corrected “SK6” for “S6K1”.

Point 16: Line 388 IRS.1

Response: We apologize there was an error in the text. We have corrected “IRS.1” for “IRS-1”.

Point 17: Lines 265-267:  please check grammar (maybe “phosphorylation that occurs” would work

Response: We very much appreciate your suggestion. We corrected line 291.

Author Response File: Author Response.pdf

Reviewer 4 Report

The presented work is built around two tables (Table 1 and Table 2), extensively collecting literature on the phosphorylation sites of serines and tyrosines in IRS1 and IRS2 substrates. The text is divided into carefully selected subsections. Indeed, the text does not describe in detail all the cited works included in Tables 1 and 2, but this seems to be a deliberate intention of the authors, whose main idea was to guide the reader through the intricate aspects of the consequences of phosphorylation of IRS factors in various sites and their consequences at the cellular and physiological level of the organisms.

Author Response

Reviewer 4

Comments and Suggestions for Authors

 Point 1: The presented work is built around two tables (Table 1 and Table 2), extensively collecting literature on the phosphorylation sites of serines and tyrosines in IRS1 and IRS2 substrates. The text is divided into carefully selected subsections. Indeed, the text does not describe in detail all the cited works included in Tables 1 and 2, but this seems to be a deliberate intention of the authors, whose main idea was to guide the reader through the intricate aspects of the consequences of phosphorylation of IRS factors in various sites and their consequences at the cellular and physiological level of the organisms.

Response: We express our gratitude to Reviewer-1 for the valuable comments on the manuscript. We fully agree with the reviewer's comment about why we decided not to describe in detail all the cited works included in Tables 1 and 2.

Point 2: The work is flawless and will be helpful as a compendium of knowledge for people looking for precise information on IRS phosphorylation sites.

Response: Thank you so much for your important comments.

Point 3: There is a small thing to improve - two dots at the end of the signature Figure 1–lane 80

Response: Thank you so much for your comment. We have deleted de extra dot from the signature in Figure 1.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have done their best to accommodate the reviewers' comments.

Reviewer 3 Report

Looks good!