1. Reviewer #3 (Public Review):

    This manuscript sheds new light on the regulation and function of a signaling network comprised of the adaptor molecules Cas and BCAR3. The data presented in the manuscript are generated through rigorous experimentation, frequently with the use of multiple approaches to arrive at the stated conclusions.

    Minor concerns:

    1. Figure 3e. The authors state that "SOCS6 binds BCAR3 and Cas independently" (bottom of page 7). However, while they show that the EE BCAR3 mutant binds to SOCS6 under conditions when it does not bind to Cas, they do not show the reciprocal interaction in this paper. Their previous paper (J Cell Sci 2014) suggests that SOCS6 binding to Cas may be independent of BCAR3 but neither that paper nor the current manuscript explicitly examine that. Unless there is direct evidence that SOCS6 can bind to Cas in the absence of BCAR3, perhaps it would be more accurate for the authors to limit their conclusion by saying that "SOCS6 binds to BCAR3 independently of Cas."

    2. Figure 8a and c. Without showing a Western blot to address total pools of phosphorylated Cas, it is not clear whether the depletion in pY165 is targeted to the pool of Cas present in adhesions or to a diminution in phosphorylation of the total pool of Cas in the cell. At a minimum, the authors would need to clarify that phosphorylation at Y165 of Cas in the pool of Cas that is localized to adhesions is reduced in the presence of Y117F, R177K, or the EE mutant of BCAR3.

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  2. Reviewer #2 (Public Review):

    This is a well-written paper describing the co-recruitment of p117-BCAR3 and Cas to adhesion sites for activation of lamellipodial ruffling and the subsequent ubiquitin-dependent degradation. The completeness of the description of the cycle is a major success of this article and warrants publication. I didn't find major holes in their arguments and they did document that this pathway was not universal but there were possibly analogous signaling processes with other players.

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  3. Reviewer #1 (Public Review):

    Summary: The study by Steenkiste focuses on the formation of adaptor protein complexes at sites of integrin receptor adhesion in the modulation of in vitro membrane ruffling and cell movement. The authors are studying the role of BCAR3 (also termed AND34 or NSP1) protein regulation by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). This is one of many adaptor proteins localized to adhesion sites. Studies are being performed on MCF10A or Hela cells to knockdown (siRNA) or over-express tagged protein constructs. By proteomics, a new phosphorylation site was identified (BCAR3 Y117). Mutagenesis showed that BCAR3 Y117 is important for enhancement of in vitro cell movement under conditions where the cullin-5 E3 ligase has also been reduced by siRNA expression.

    Opinion: The authors provide support for a "co-regulatory" model whereby the recruitment of BCAR3 to adhesions acts in part to modulate another adaptor protein tyrosine phosphorylation, p130Cas. This is associated with enhanced cell migration. The data presented are generally supportive of the conclusions and consistent with previous studies of BCAR3 and p130Cas. However, an unresolved issue is why cell phenotypes are dependent on cullin-5 knockdown or otherwise investigated by BCAR3 mutant over-expression. Cul5 loss can alter multiple aspects of cell signaling and the transient knockdown or inducible over-pression assays are a limited primary means of investigation. As multiple protein domains and post-translational modifications modulate the BCAR3-p130Cas complex, the authors did not establish a strong mechanistic linkage between newly-identified BCAR3 Y117 phosphorylation, SOCS6 binding, and a CUL5-dependent cell phenotype. Additionally, some of the experimental conditions (+/- EGF in growth media) are difficult to connect to EGF receptor activation and or signaling.

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  4. Evaluation Summary:

    This study focuses on the formation of adaptor protein complexes at adhesion sites and their links to in vitro membrane ruffling and cell movement. Specifically, the authors study the role of the adaptor BCAR3 protein which is regulated by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). The authors propose a "co-regulatory" model whereby the recruitment of BCAR3 to adhesions acts to modulate p130Cas tyrosine phosphorylation and cell migration. This manuscript would be of particular interest to cell and cancer biologists interested in the molecular regulation of cell migration.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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