Peer review of: AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion (2020)



Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript is improved over the previous version, but a few issues remain to be resolved 
before the work is complete 

1) The method of quantifying PLA signal seems odd (total average intensity versus number of 
puncta). The method of analysis does not tell you how much more PLA signal is there when an 
interaction is detected? 
2)Figure 7 still seems a mess to me. Many of the blots are blown out/have poor exposures, and 
the graphs of FA lifetime have no primary data, such as the examples presented in Fig 3. 

Reviewer #2 (Remarks to the Author): 

The authors miss the point of the Gas6 issue. Yes, gamma-carboxylation is required for Gas6-
mediated AXL activation but so is association of Gas6 with PS lipids -- indeed, the role of gamma-
carboxylation is to facilitate Gas6/PS association. This has been established by multiple literature 
reports from a variety of laboratories. The extent to which Gas6 obtained from one source or 
another is gamma-carboxylated is relevant only in terms of its interaction with PS. Thus, the 
presence of PS lipids, such as from apoptotic cellular debris or other conditions, in the 
experimental assay cultures is critical in governing AXL activation, regardless of the extent of Gas6 
gamma-carboxylation. Now, if one has a source of Gas6 that is more extensively gamma-
carboxylated than a different source, the amount of PS lipids needed to be present in the assay 
environment to obtain a given level of AXL activation may be lesser, but Gas6 by itself cannot 
activate AXL to an appreciable degree even if extensively gamma-carboxylated. 

Reviewer #3 (Remarks to the Author): 

The authors have clarified my previous concerns and have addressed previous points/concerns 
raised by carrying out further experiments. 

Reviewer #4 (Remarks to the Author): 

The manuscript by Afnan Abu-Thuraia, et al investigated the signaling mechanisms mediated by 
AXL, one of the receptor tyrosine kinases (RTKs) that are activated during metastasis of breast 
cancer. Using quantitative phosphoproteomics, the authors identified PEAK1 as a critical 
downstream factor of the AXL activation and PEAK1 promoted the turnover of focal adhesion (FA) 
by recruiting the FA turnover complex to paxillin. Knockout of PEAK1 inhibited breast cancer cell 
metastasis, which can be rescued by wild-type PEAK1 but not mutant PEAK1 that lost interaction 
with the pathway. 
Overall, the study reported an interesting finding on the important role of PEAK1 in mediating the 
activity of FA turnover upon AXL activation. However, a number of key mechanisms are not clear 
despite the observation of AXL-mediated phosphorylation events. In addition, the logic and study 
design of the overall study described in the manuscript was quite complicated and not all 
experiments were necessary to be included in the manuscript. Some major and minor concerns are 
listed below. Due to these concerns, the study is not yet ready for publication in its current form. 
1. Identification of PEAK1 was achieved by overlapping the phosphoproteomics data of AXL 
activation with the interactome data of NEDD9. However, it is quite strange that the interaction 
between NEDD9 and PEAK1 could not be validated (line 265). This cast doubts on the validity of 

the NEDD9 interactome analysis with Bio-ID method. 
2. The mechanism of how AXL activates PEAK1 was still not clear. Since the interaction between 
PEAK1 and AXL was validated and then PEAK1 phosphorylation increased upon AXL activation, the 
authors hypothesized that PEAK1 was a direct AXL target. However, mutation of the identified 
tyrosine residues did not inhibit AXL-mediated PEAK1 phosphorylation. So the key question is that 
whether PEAK1 phosphorylation was required for its activation. It is also possible that AXL 
activates PEAK1 indirectly or through protein-protein interactions. 
3. For validation of AXL control of PEAK1 localization at FA, the authors applied siRNA and chemical 
treatment to knock down or inhibit AXL activity. However, these experiments are not sufficient to 
demonstrate the importance of AXL kinase activity in this process. Activation of AXL and kinase 
dead mutant by overexpression and GAS6 treatment would be more informative to determine if 
PEAK1 phosphorylation by AXL plays an important role during this process. 
4. The overall role of NEDD9 in PEAK1-mediated phosphorylation and activation of FA turnover is 
not very clear. The manuscript included Figure 4 and a large section of texts describing the 
validation and interaction of NEDD9 with FA. However, such information only led to the 
identification of PEAK1 and not quite relevant with the key findings of the current manuscript. 
Therefore, the inclusion of these data on NEDD9 made it difficult to follow the logic flow of the 
study. 
5. The mechanisms on how AXL-mediated CRKII phosphorylation regulates its interaction with PXN 
was not clear. Since the AXL-mediated phosphorylation sites on CRKII have been identified, it 
would be straightforward to test if these phosphorylation events are important for CRKII 
interaction with PXN. 
6. The importance of PEAK1-mediated phosphorylation of PXN was not clear. The authors showed 
that PEAK1 may be able to directly phosphorylate PXN and it was also possible that CSK through 
PRAG phosphorylates PXN. These possibilities were not rigorously studied and should not be 
included if the findings are not conclusive. It is also unclear what may be the site-specific effect of 
PEAK1-mediated PXN phosphorylation in FA turnover. 
7. The interaction between PEAK1 and PAK1 complex was identified and the authors hypothesized 
that these interactions are important for FA turnover. However, other than the knockdown study, 
there is no further mechanistic study to prove the hypothesis. 
8. The authors demonstrated the important role of PEAK1 and CRKII complex in FA turnover using 
3PA PEAK1 mutant. However, the key question is whether AXL mediates the interaction between 
PEAK1 and CRKII. 
9. Since 3PA PEAK1 mutant is the key to demonstrate the important role of activation of PEAK1 by 
AXL in vivo, it is important to determine if the mutation affect the interaction between PEAK1 and 
b-PIX/GIT1/PAK1 complex. 
10. The proteomics data analysis figure (Figure 1c) is confusing. For quantitative proteomics data, 
please perform three biological replicates for each time point and use volcano plots to identify 
significantly changed hits. 
Minor concern: 
1. Figure 1d, how the relative abundance of phosphorylation was defined? Why was the 0 min 
abundance for each site not shown? The figure may be better presented as line graphs. 
2. Figure 2 can be put in Supplementary material as the analysis does not contribute significantly 
to identification of PEAK1 and other targets. 

Reviewer #5 (Remarks to the Author): 

The revised manuscript by Abu-Thuraia et al., addresses both the scientific concerns and the 
issues raised by reviewers with the presentation of the proposal during the prior cycle of review. 
The manuscript addresses the potential role of an AXL/PEAK regulated signaling axis consortium in 
promoting cell adhesion and cell migration. The work provides a new rationale for the regulation of 
cell motility downstream of AXL via CrkII and NEDD9, which may be important not only in 

pathologies such as cancer, but also during development. The authors have done a good job of 
addressing most of the prior issues. There are still a couple issues related to prior points which 
should be addressed: 

The authors conclude that PEAK1/CrkII interaction is required for “localization” at FAs. It remains 
possible that PEAK1 is not responsible for localization/targeting, but acts to stabilize CrkII within 
the FAs. This should be addressed within the text, at a minimum. 

The studies are highly dependent upon cell lines and upon Axl over-expression, and this seems 
partly due to a lab-constructed system. Axl is expressed in only a fraction of invasive breast 
cancers and it is a stretch to claim it is “highly expressed.” In fact, Axl is a significant negative 
prognostic only in renal cancer (where PEAK1 is abundant). In other tissues, which express high 
levels of both PEAK1 and Axl, (such as testicles, where co-expression is the highest) Axl 
expression is typically lost during oncogenesis. The authors should address this dichotomy in order 
to provide their results in a correct context. 

Minor: The presentation of the bioinformatics includes gratuitous graphics (which are generally 
intended for the presentation of much bigger data matrixes) (eg., Figure 2a, d). In these cases, a 
simple table would provide more information to the reader while occupying less space.