SHARPIN S146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion and metastasis

  1. Umar Butt
  2. Meraj H. Khan
  3. Jeroen Pouwels
  4. Jukka Westermarck

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

SHARPIN is involved in several cellular processes and promotes cancer progression. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here, we characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on S131 and S146, we demonstrate that they have a role in SHARPIN-ARP2/3 complex interaction, but play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, S146 phosphorylation of SHARPIN promoted lamellipodia formation. We also demonstrate that SHARPIN S146 phosphorylation-mediated ARP2/3 interaction is sensitive to inhibition of ERK1/2 or reactivation of protein phosphatase 2A (PP2A). Notably, CRISPR/Cas9-mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces in vivo metastasis in a zebrafish model. Collectively, these results map SHARPIN phosphorylation sites and identify S146 as a novel phosphorylation switch defining ARP2/3 interaction and cancer cell invasion.

This article has an associated First Person interview with the first author of the paper.

Article activity feed

  1. Version published to 10.1242/jcs.260627
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Comments for Butt et al This study examines the potential role of SHARPIN phosphorylation on cell migration. The role of the phosphorylation sites of SHARPIN, especially S146, is clearly shown by the expression of the mutant in various cell lines. However, the argument of the interaction with the Arp2/3 complex is relatively weak; only the FRET analysis in cells is shown. It would be good if the authors could include more mechanistic insights into the role of the phosphorylation of SHARPIN.

    Table 1. the reason for the selection of S131 and S156 is not clear. Why the S165 and T170 were not studied?

    The phosphorylation would be better confirmed by the antibody for the phosphorylation peptides. The possible phospho-mimic mutants, with the substitution to the acidic amino acid residues, would be better to be examined.

    Significance

    The potential role of SHAEPIN in the context of the current understanding of the cell migration machinery would be better to be described. Now the study only contains the mutant expression phenotype.

    Referees cross-commenting

    I agree with other reviewers and do not have other opinions. The three reviews appear to be reasonable.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this paper, Butt and co-workers examine the role of SHARPIN phosphorylation in Ser146 as it controls diverse readouts related to cell motility. The main findings of the study are: SHARPIN is robustly phosphorylated by PKCalpha in vitro. Mass spectrometry and in silico approaches indicate that some of these sites may be genuinely phosphorylated in cells. Ser146 phosphorylation seems uninvolved in SHARPIN-mediated integrin inactivation but imitation of the non-phosphorylated state using a Ser to Ala mutant impairs SHARPIN interaction with Arp2/3. S146A and V240A/L242A mutations impair lamellipodia formation. S146E mutation has a much less pronounced effect on SHARPIN-Arp2/3 interaction and lamellipodia formation, being close to the effect of the wild type. The authors conclude, based on this, that the active form of SHARPIN is likely phosphorylated in Ser146. In 3D matrix invasion assay, CRISPR-mediated SHARPIN deletion decreased invasiveness in several tumor cell lines. Perhaps the most striking experiment is that the authors use SHARPIN-KO MDA-MB-231 cells reconstituted with GFP-SHARPIN, WT or 146A, in a xenograft model in zebrafish, and they see that only WT-expressing cells form distal clusters of tumor cells. This is an interesting paper that merits publication, but several issues need to be addressed.

    1. Whereas the data are consistent and interesting, some points could be strengthened quantitatively. An example is the quantification of the invasion assays (Fig. 4), which is relatively crude (this reviewer has firsthand experience with these assays).
    2. Does SHARPIN control actin polymerization in response to stimulation? For example, growth factors in the cells used, or PMA.
    3. PKCalpha has been involved in the control of protrusion dynamics through its local effect on myosin II regulatory light chain (Asokan et al, 2014). Is PKCalpha actually phosphorylating SHARPIN in live cells? Are other kinases involved in vitro? This needs to be clearly and explicitly demonstrated.
    4. SHARPIN phosphorylated in Ser146 locally at lamellipodia? This is a hard experiment that requires a phospho-specific antibody, but the subcellular localization of the effect may be critical towards explaining the relative importance and generality of this mechanism.
    5. In the same vein, does SHARPIN S146E localize more readily to protrusions that Ser146A?
    6. The inference that active SHARPIN is phosphorylated in Ser146 needs to be demonstrated formally for the story to be substantiated. One possible manner could be to immunoprecipitate the Arp2/3 complex and show that most of the associated SHARPIN is phosphorylated in this residue by mass spectrometry. Alternatively, the authors could pull down integrins and show that SHARPIN is not phosphorylated in this case, which is also suggested by their data.
    7. The last piece of data is striking, but the xenograft nature of the experiment casts some doubt as to its significance. B16F10 cells similarly treated could be implanted in C57BL/6 mice to make a much stronger case.

    Significance

    The data presented here, if substantiated as indicated in the previous section, would constitute a significant addition to the state of the art. My enthusiasm for the story is curbed by the technical flaws (relatively minor) and conceptual gaps (more significant) stated above. My expertise is cell and molecular biology with focus on cytoskeletal-related cell motility.

    Referees cross-commenting

    I mostly concur with the revisions suggested by the other two reviewers. Reviewer #1 raises a significant point that needs to be addressed, which pertains to the overall levels of overexpression. Other than that, I stand by my review, which has many connection points with those of reviewers 1 and 3. I think this paper has been judged fairly and the experiments requested are not overly complicated. I understand if the authors cannot do the in vivo experiment in mice, and this alone should not be grounds for rejection. However, they need to address the rest of our points.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The SHARPIN protein is involved in multiple cellular pathways, and a number of studies demonstrated that it can interact with many key proteins regulating cellular proliferation, adhesion, motility and other functions that are important in normal cell physiology, but are particularly relevant for cancer metastasis. This study addresses post-translational phosphorylation of SHARPIN in human cells, and identifies two functionally important sites that are specifically involved in the interaction with ARP2/3, as well as in lamellipodia formation and cell motility (invasion). The function of one of these phosphorylation sites, the S146, is further explored in KD, KO and rescue experiments, in several human cancer cell lines, and in zebrafish model, using the directed mutagenesis approach to abolish or mimic the phosphorylation of S146. The authors conclude that Ph-S146 modification of SHARPIN is specifically responsible for interaction with ARP2/3, lamellipodia formation, and cancer cell invasion.

    Major comments:

    • Are the key conclusions convincing?

    Overall, the conclusions are convincing, though the question of wild-type and mutated SHARPIN in rescue experiments should be addressed, given the functional importance of SHARPIN overexpression in cancer. Indeed, throughout the paper, the authors monitor the expression levels of their ectopically expressed SHARPIN, and systematically refer to these molecules as being "overexpressed", without showing their relative levels with regard to normal endogenous levels of SHARPIN in these cell lines, prior to its KD/KO. However, as the typical cancer-related functions of SHARPIN are linked to its expression levels, it is important to understand whether the observed phenomena can be regarded as physiologically relevant, or are the authors operating out of the physiological scale. Given the capacity of HeLa and 293 cell lines to produce very high levels of ectopic proteins, this issue should be systematically controlled. Therefore, in all the experiments with ectopic expression of SHARPIN and its mutants, a western blot should be added to show the relative expression, compared to the endogenous protein.

    There are also some major problems with the statistical analysis (please see below).

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    I would modify the text of the article referring to Fig.2A, where the authors qualify a 20-25% reduction of integrin activity as "significant". It is not clear whether they refer to statistical or functional significance, and the statement is generally misleading.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    As mentioned previously, I believe that the expression levels of ectopic SHARPIN have to be systematically monitored in all assays, and compared to the normal, endogenous levels.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    These experiments are fully realistic and should not involve additional costs.

    • Are the data and the methods presented in such a way that they can be reproduced?

    There is some ambivalence about the "n" values in most of the experiments, where it is not clear at all what "n" means (the number of cells? Experimental points?) For example, in Fig.S1D, the legend states that n=1, whereas the panel shows three experimental points per condition, as well as error bars. I would suggest that the authors correct these obvious mistakes and explain more clearly what exactly "n" means, in each case.

    • Are the experiments adequately replicated and statistical analysis adequate?

    I cannot comment on that because I do not understand what exactly "n" means (please see above). For example, if in Fig.3, "n=4" means that only four cells were analyzed per experimental condition, this is clearly not enough. The statistical analysis is not sufficiently described in the Materials and Methods section, and the reasons for attributing one, two or three stars to the results are not stated, either (normally, this information should be equally present in Legends to Figures). The choice of applying the t-test does not appear evident to me, in experiments that clearly require multiple statistical comparisons, and in general, the statistical analysis does not appear adequate.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Please provide a clear gel for Fig.1A, especially the right-hand panel. The current results look as one big black spot, with two red frames added for no clear reason.

    • Are prior studies referenced appropriately?

    I have no problem with the list of references.

    • Are the text and figures clear and accurate?

    It will be a good idea to proof-read the text. For example, I have noticed a frequent use of the word "lamellApodia" instead of "lamellipodia", as well as other typing errors.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Molecular weight markers should be added to all western blots, and scale bars to all immunofluorescent images.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Given the functional importance of SHARPIN in cancer, and the fact that it interacts with multiple major regulatory networks, it is important to pinpoint the exact post-translational modification of this protein that is specifically responsible for interaction with ARP2/3, and for the invasion potential of cancer cells.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    I agree with the description of the field and the place of the current study that is provided in the manuscript, and do not have anything significant to add.

    • State what audience might be interested in and influenced by the reported findings.

    The study will undoubtedly be interesting to scientists working with cell motility, ARP2/3-dependent lamellipodia formation, and eventually metastasis growth in cancer.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I study the RAC1-WAVE-ARP2/3 regulatory pathway in normal and cancer cells. I did not have any problems with evaluating this work, either academically, or with regard to methodology.

    Referees cross-commenting

    I am very pleased to see that the three reviews have a very similar evaluation of this article, and hope that the raised questions will help the authors to improve the manuscript and successfully publish their work. I do not have any additional comments.

  6. Version published to 10.1101/2022.01.21.477220v1 on bioRxiv