The Axin scaffold protects the kinase GSK3β from cross-pathway inhibition

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1 (Recommendations for the authors):

– At the start of the Results section and in Figure 2, the definitions of kCAT and Km should be given in a bit more detail (at present the reader is only provided with a reference to the 2020 paper). This will make the story a bit easier to read for non-biochemists. For example, lines 106-109 are not intuitive to follow.

We have added additional explanation of the significance of k_cat and K_M values at the start of the Results section (lines 61-62). We have also added content and a reference to clarify lines 106-109 (now lines 83-86). Given the space restrictions for the Short Report format our explanations are brief, we request editorial guidance if further elaboration is necessary.

– The added value of the kinetic schemes in each of the figure panels is unclear as the link to specific biological/signal transduction steps is not made clear.

Kinetic schemes are included with each figure panel to enable clear comparisons between different reactions. We appreciate the reviewer’s point that it would be helpful to connect each scheme to a biological step, but we suggest that significant space would be necessary to place each reaction in a biological context and would largely reproduce information that is available in Figure 1.

– The cartoons in Figure 1 could also be improved to clarify the questions/knowledge gaps and specific mechanistic options.

We have added a panel to Figure 1 to clarify the mechanistic models and we have corrected a mistake that was noted by Reviewer #2. We would be happy to further revise the figure if the editors or reviewers have additional suggestions.

– The authors convincingly show that AXIN1 can play a role in shielding GSK3 from auto- inhibition. As it stands, the impact of this work on the field of WNT/CTNNB1 signaling is likely to remain limited. This is mainly due to the reason that the mechanism by which AXIN1 shields the WNT/CTNNB1 signaling pool of GSK3 from pSer9 inhibition remains unresolved. Based on the fact that a mini AXIN1 (i.e. an AXIN1 fragment) behaves the same as WT AXIN1, the authors conclude that AXIN1 likely causes allosteric changes on GSK3 but is less likely to block PKA from binding. They cannot conclusively show this, however, as they do not have evidence in favour of one or the other explanation.

We thank the reviewer for this important comment which details the central concern raised in the review process. To address this point, we have collected additional biochemical data that conclusively shows that the Axin shielding effect is allosteric and not a steric block. We demonstrated that a minimal, 27 amino acid Axin peptide produces the same GSK3β shielding behavior as full length Axin and miniAxin. The minimal Axin peptide does not sterically occlude the GSK3β phosphorylation site. This data is included in a revised Fig 4A and described on lines 151-157 of the revised manuscript.

– The impact of this work would be greater if the authors could indeed discriminate between the two possible mechanisms they present (allosteric changes on GSK3 or blocking the binding of PKA).

We agree with the reviewer on this central point and have conducted additional experiments that conclusively resolve the issue. These experiments are described above and included in the revised manuscript (Figure 4A, lines 115-120).